The Effect Of Polarization Of Macrophage Upon Healing Of Tooth Extraction Socket Under T2D

[Objective] Healing of tooth extraction is often delayed in patients suffered from poor-controlled type 2 diabetes(T2D)and infection is considered as an important reason,however,the occurrence mechanism of inflammation is unknown.In the present study,we firstly conduncted the investigation of healing of tooth extraction socket under type 2 diabetes mellitus.Secondly,in-vitro determination was to verify the effects of different polarized BMDMs upon the osteogenic differentiation of BMSCs.And then,loaded IL-4 hybrid scaffold was fabricated to intervene the immune environment of extraction socket in diabetic mice and promoted the healing process.This study provided the experimental evidence for exploring a potential treatment for impaired bone regeneration under type 2 diabetic mellitus.[Methods]Part 1.The study of macrophage characterization and healing process of extraction socket in type 2 diabetic mouse :(1)Combination of high-fat diet(HFD)and streptozotocin(STZ)was to induce experimental type 2 diabetic mouse;(2)After the induction of animal model,surgery of tooth extraction was conducted and animal samples were harvested at 7 days and 28 days post-extraction for further determination;(3)Means of radiography and histology were performed to analyzethe osteogenic differentiation ability,inflammatory environment and polarization of BMDMs.Part 2.The study of effect of macrophage polarization upon osteogenic differentiation of BMSCs in-vitro:(1)M0,M1 and M2 macrophages were respectively cocultured with BMSCs for 14 days;(2)Real-time PCR was used to detect the expression of Runx2,ALP,OSX and OCN;(3)Content of Pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 was detected by the method of ELISA;(4)After the treatment of different concentration of TNF-α cytokines(0,5,10,20ng/ml)on BMSCs for 72 hours,western blot was performed to analyze the protein expression of OPN,Runx2 and OSX.Part 3.The promotion of macrophage polarization by the releasement of IL-4from Gelatin/β-TCP scaffold :(1)Fabrication of Gelatin/β-TCP scaffold loading IL-4 with different content of β-TCP;(2)Scanning electron microscope was used to detected the micro-structure of Gelatin/β-TCP scaffold unloading IL-4 with 0% and25% content of β-TCP;(3)M0 macrophages were cocultured with Gelatin/β-TCP scaffold loading or unloading IL-4 with 25% content of β-TCP and western blot was performed to analyze the expression of CD86 and CD206 in macrophages;(4)ELISA was used to detected the content of TNF-α and IL-10 in supernatant of BMDMs.Part 4.The promotion of extraction socket healing by the releasement from Gelatin/β-TCP scaffold in type 2 diabetic mouse :(1)After surgery of tooth extraction,loaded or unloaded IL-4 Gelatin/β-TCP scaffold was immediately implanted into socket and the sample was harvested respectively at 7 days and 28 days post-extraction;(2)Radiography determination was used to analyze the healing of extraction socket;(3)Osteogenic differentiation ability,inflammatoryenvironment and polarization of macrophage of extraction socket was detected by means of immune histology.[Results]Part 1: The study of macrophage characterization and healing process of extraction socket in type 2 diabetic mouse:(1)Healing of tooth extraction socket in mice is delayed under type 2 diabetes mellitus;(2)Type 2 diabetes mellitus led to the impaired osteogenic differentiation ability in extraction socket;(3)Type 2 diabetes mellitus led to chronic inflammation in extraction socket;(4)A abundant of M1 macrophages recruited in extraction socket during all the period of healing process under type 2 diabetes mellitus and the swift of M1 to M2 phenotype was delayed.Part 2: The study of effect of macrophage polarization upon osteogenic differentiation of BMSCs in-vitro:(1)Osteogenic differentiation ability of BMSCs was suppressed in coculture system with M1 macrophages;(2)More TNF-αcytokine in supernatant of BMSCs was secreted from M1 macrophage in upper chamber;(3)More IL-10 cytokine in supernatant of BMSCs was secreted from M2 macrophage in upper chamber;(4)TNF-α suppressed osteogenic differentiation ability of BMSCs at a certain dosement and time manner.Part 3: The promotion of macrophage polarization by the releasement of IL-4 from Gelatin/β-TCP scaffold :(1)IL-4 cytokine released consistently from Gelatin/β-TCP scaffold consistently;(2)As content of gelatin and method of crosslink was equal,the retention of IL-4 was not interfered with the β-TCP content;(3)Releasement of IL-4 from Gelatin/β-TCP scaffold induced polarization of M2 macrophages;(4)Releasement of IL-4 from Gelatin/β-TCP scaffold suppressed polarization of M1macrophages;(5)Releasement of IL-4 from Gelatin/β-TCP scaffold promoted secretion of anti-inflammatory IL-10 cytokine from M2 macrophages;(6)Releasement of IL-4 from Gelatin/β-TCP scaffold suppressed secretion of pro-inflammatory TNF-α cytokine from M1 macrophages.Part 4: The promotion of extraction socket healing by the releasement from Gelatin/β-TCP scaffold in type 2 diabetic mouse:(1)Gelatin/β-TCP+IL-4 scaffold promoted the healing of tooth extraction socket in type Ⅱ diabetic mouse;(2)Gelatin/β-TCP+IL-4 scaffold promoted the osteogenic differentiation ability of tooth extraction socket in type Ⅱ diabetic mouse;(3)Gelatin/β-TCP+IL-4 scaffold suppressed chronic inflammation in tooth extraction socket of type Ⅱ diabetic mouse;(4)Gelatin/β-TCP+IL-4 scaffold promoted the shift from M1 to M2 macrophage in tooth extraction socket of type Ⅱ diabetic mouse.[Conclusion] 1.In tooth extraction socket,type Ⅱ diabetes mellitus resulted to the recruitment of M1 macrophages,chronic inflammation and suppression of osteogenic differentiation ability,eventually socket healing was retarded;2.In-vitro,M1 macrophages suppressed the osteogenic differentiation of BMSCs;3.Polarization of M2 macrophage was induced but M1 macrophage was suppressed by IL-4 released from Gelatin/β-TCP scaffold;4.Under type Ⅱ diabetes mellitus,the shift from M1 to M2 macrophages in extraction socket was induced by the releasement of IL-4 from Gelatin/β-TCP scaffold,sequentially,local prolonged inflammation was suppressed,osteogenic differentiation and socket healing was promoted.