3D打印仿生鈦骨表面復(fù)合涂層對(duì)成骨細(xì)胞相容性及抑菌性能的研究
發(fā)布時(shí)間：2018-08-09 14:16
【摘要】：目的:應(yīng)用3D打印技術(shù)制作具有三維孔隙相互通連結(jié)構(gòu)的仿生鈦骨,分別復(fù)合殼聚糖-甘油磷酸溫敏水凝膠涂層和殼聚糖納米粒兩種生物活性涂層,觀察仿生鈦骨及不同涂層表面對(duì)成骨細(xì)胞黏附、增殖和分化的影響以及對(duì)牙齦卟啉單胞菌的抑菌效果。方法:3D打印制作孔隙大小為300um的仿生鈦骨試件;將殼聚糖溶解在乙酸溶液中與甘油磷酸溶液按8:2混合形成殼聚糖-甘油磷酸溫敏水凝膠(CS/α,β-GP),與三聚磷酸鈉(TPP)溶液按10:1混合形成殼聚糖納米粒(CSn)。實(shí)驗(yàn)分組:Ti:無涂層的仿生鈦骨組;Ti+CS/GP:復(fù)合CS/α,β-GP涂層的仿生鈦骨組;Ti+CSn:復(fù)合CSn涂層的仿生鈦骨組;Contr:空白孔板組,作為陽性對(duì)照。⑴.SE M觀察各組3D打印仿生鈦骨的結(jié)構(gòu)形態(tài);⑵.體外培養(yǎng)MC3T3-E1細(xì)胞,并接種于不同涂層仿生鈦骨上,熒光技術(shù)觀察細(xì)胞的黏附情況,CCK-8法檢測(cè)細(xì)胞的增殖,ALP試劑盒檢測(cè)細(xì)胞的分化狀況;⑶.體外培養(yǎng)牙齦卟啉單胞菌,細(xì)菌培養(yǎng)計(jì)數(shù)法檢測(cè)其在不同生物活性涂層仿生鈦骨上的黏附情況。所得數(shù)據(jù)均采用SPSS18.0統(tǒng)計(jì)軟件處理。結(jié)果:⑴.3D打印仿生鈦骨表面為尺寸一致的網(wǎng)格狀結(jié)構(gòu),孔隙分布均勻,相互貫通;內(nèi)部多孔隙結(jié)構(gòu)能夠促進(jìn)血液快速附著,增強(qiáng)血塊穩(wěn)定,縮短種植愈合時(shí)間。掃描電鏡顯示:3D打印仿生鈦骨呈均勻分布的網(wǎng)格狀結(jié)構(gòu),網(wǎng)格間交匯相通;CS/α,β-GP及CSn在仿生鈦骨上均勻分布。⑵.熒光顯微鏡顯示:MC3T3-E1在各組試件表面及孔隙內(nèi)黏附生長;Contr組胞體突觸伸展呈多角型,可見細(xì)長的突觸,Ti+CSn組與Ti+CS/GP組細(xì)胞黏附在鈦骨表面,胞體伸展呈類圓形,突觸伸展呈多角型,但觸角伸展不明顯,Ti組胞體呈類圓形,無明顯的多角形態(tài);MC3T3-E1接種后分別培養(yǎng)1、3、5、7天,CCK-8檢測(cè)顯示:ContrTi+CSnTi+CS/GPTi,各組間存在明顯統(tǒng)計(jì)學(xué)差異;ALP檢測(cè):Ti+CSn組與Ti+CS/GP組的ALP分泌均高于Ti組,存在明顯的統(tǒng)計(jì)學(xué)差異;Contr組的ALP分泌均低于Ti+CSn組、Ti+CS/GP組,具有顯著的統(tǒng)計(jì)學(xué)差異。⑶.細(xì)菌黏附實(shí)驗(yàn)結(jié)果顯示:Ti+CS/GP組、Ti+CSn組表面細(xì)菌黏附數(shù)均明顯少于Ti組(P0.05),且Ti+CSn組Ti+CS/GP組(P0.05)。結(jié)論:(1)3D打印仿生鈦骨有利于血液快速的進(jìn)入仿生鈦骨內(nèi),促進(jìn)血液在表面的附著,增強(qiáng)血塊穩(wěn)定性,促進(jìn)骨血管化,縮短骨結(jié)合時(shí)間;(2)3D打印仿生鈦骨結(jié)構(gòu)具有良好的細(xì)胞相容性,復(fù)合殼聚糖納米粒涂層后更有利于成骨細(xì)胞早期的黏附生長、增殖及成骨分化;(3)殼聚糖涂層可以有效抑制牙齦卟啉單胞菌的黏附;殼聚糖納米粒抑菌效果優(yōu)于殼聚糖-甘油磷酸溫敏水凝膠。
[Abstract]:Objective: to fabricate bionic titanium bone with three-dimensional porous interconnecting structure by using 3D printing technology, and composite chitosan glycerophosphoric acid thermo-sensitive hydrogel coating and chitosan nanoparticles bioactive coating, respectively. The effects of biomimetic titanium bone and different coatings on the adhesion proliferation and differentiation of osteoblasts and the bacteriostatic effect on porphyromonas gingivalis were observed. Methods the biomimetic titanium bone specimens with pore size of 300um were made by using 30% 3D printing. Chitosan was dissolved in acetic acid solution and mixed with glycerophosphoric acid solution at 8:2 to form CS/ 偽, 尾 -GP, and then mixed with sodium tripolyphosphate (TPP) solution to form chitosan nanoparticles (CSn). At 10:1 The experiment was divided into two groups: TICs / GP: CS/ 偽, 尾 -GP coated bionic titanium bone group: CSn coating bionic titanium bone group: blank hole plate group, as positive control. 1. SE M was used to observe the structure and morphology of 3D printed biomimetic titanium bone in each group. MC3T3-E1 cells were cultured in vitro and inoculated on different coating biomimetic titanium bone. The cell adhesion was observed by fluorescence technique and the proliferation of MC3T3-E1 cells was detected by CCK-8 method. Porphyromonas gingivalis was cultured in vitro. The adhesion of Porphyromonas gingivalis on biomimetic titanium bone with different bioactive coatings was detected by bacterial culture counting method. All the data were processed by SPSS18.0 software. Results: 1. The surface of the biomimetic titanium bone was of uniform size, the pore distribution was uniform, and the porous structure could promote the rapid blood adhesion, enhance the stability of blood clot and shorten the time of implant healing. Scanning electron microscope (SEM) showed that: 3D printed bionic titanium bone showed a uniform distribution of mesh-like structure, and the intermeshes of CSA / 偽, 尾 -GP and CSn were evenly distributed on the biomimetic titanium bone. Fluorescence microscope showed that the cell body synaptic extension of the Contr group was polygonal on the surface and pore of the specimens. The elongated cells of Ti CSn group and Ti CS/GP group adhered to the surface of titanium bone, and the cell body stretched round. The synaptic extension was polygonal, but the cell bodies in the Ti group were round, but the antennal extension was not obvious. After inoculation with MC3T3-E1 without obvious polygonal morphology, CCK-8 was detected in 10% ContrTi CSnTi CS-GPTi. there was significant statistical difference in ALP secretion between the two groups. The ALP secretion in the two groups was higher than that in the Ti CS/GP group, and the ratio of ALP secretion between the two groups was higher than that in the Ti group. The ALP secretion in Contr group was significantly lower than that in Ti CSn group and Ti CS/GP group, and there was significant statistical difference between Contr group and Ti CS/GP group. The results of bacterial adhesion test showed that the number of bacteria adhesion in Ti CSn group was significantly lower than that in Ti group (P0.05), and that in Ti CSn group was significantly lower than that in Ti CS/GP group (P0.05). Conclusion: (1) 3D printing of the biomimetic titanium bone is beneficial to the rapid entry of blood into the biomimetic titanium bone, promoting the blood adhesion on the surface, enhancing the stability of the blood clot, and promoting the vascularization of the bone. (2) 3D printing biomimetic titanium bone structure has good cell compatibility, the composite chitosan nanoparticles coating is more conducive to the early adhesion growth of osteoblasts. Proliferation and osteogenic differentiation; (3) chitosan coating could effectively inhibit the adhesion of Porphyromonas gingivalis; chitosan nanoparticles had better bacteriostatic effect than chitosan glycerophosphoric acid thermo-sensitive hydrogels.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.1
[Abstract]:Objective: to fabricate bionic titanium bone with three-dimensional porous interconnecting structure by using 3D printing technology, and composite chitosan glycerophosphoric acid thermo-sensitive hydrogel coating and chitosan nanoparticles bioactive coating, respectively. The effects of biomimetic titanium bone and different coatings on the adhesion proliferation and differentiation of osteoblasts and the bacteriostatic effect on porphyromonas gingivalis were observed. Methods the biomimetic titanium bone specimens with pore size of 300um were made by using 30% 3D printing. Chitosan was dissolved in acetic acid solution and mixed with glycerophosphoric acid solution at 8:2 to form CS/ 偽, 尾 -GP, and then mixed with sodium tripolyphosphate (TPP) solution to form chitosan nanoparticles (CSn). At 10:1 The experiment was divided into two groups: TICs / GP: CS/ 偽, 尾 -GP coated bionic titanium bone group: CSn coating bionic titanium bone group: blank hole plate group, as positive control. 1. SE M was used to observe the structure and morphology of 3D printed biomimetic titanium bone in each group. MC3T3-E1 cells were cultured in vitro and inoculated on different coating biomimetic titanium bone. The cell adhesion was observed by fluorescence technique and the proliferation of MC3T3-E1 cells was detected by CCK-8 method. Porphyromonas gingivalis was cultured in vitro. The adhesion of Porphyromonas gingivalis on biomimetic titanium bone with different bioactive coatings was detected by bacterial culture counting method. All the data were processed by SPSS18.0 software. Results: 1. The surface of the biomimetic titanium bone was of uniform size, the pore distribution was uniform, and the porous structure could promote the rapid blood adhesion, enhance the stability of blood clot and shorten the time of implant healing. Scanning electron microscope (SEM) showed that: 3D printed bionic titanium bone showed a uniform distribution of mesh-like structure, and the intermeshes of CSA / 偽, 尾 -GP and CSn were evenly distributed on the biomimetic titanium bone. Fluorescence microscope showed that the cell body synaptic extension of the Contr group was polygonal on the surface and pore of the specimens. The elongated cells of Ti CSn group and Ti CS/GP group adhered to the surface of titanium bone, and the cell body stretched round. The synaptic extension was polygonal, but the cell bodies in the Ti group were round, but the antennal extension was not obvious. After inoculation with MC3T3-E1 without obvious polygonal morphology, CCK-8 was detected in 10% ContrTi CSnTi CS-GPTi. there was significant statistical difference in ALP secretion between the two groups. The ALP secretion in the two groups was higher than that in the Ti CS/GP group, and the ratio of ALP secretion between the two groups was higher than that in the Ti group. The ALP secretion in Contr group was significantly lower than that in Ti CSn group and Ti CS/GP group, and there was significant statistical difference between Contr group and Ti CS/GP group. The results of bacterial adhesion test showed that the number of bacteria adhesion in Ti CSn group was significantly lower than that in Ti group (P0.05), and that in Ti CSn group was significantly lower than that in Ti CS/GP group (P0.05). Conclusion: (1) 3D printing of the biomimetic titanium bone is beneficial to the rapid entry of blood into the biomimetic titanium bone, promoting the blood adhesion on the surface, enhancing the stability of the blood clot, and promoting the vascularization of the bone. (2) 3D printing biomimetic titanium bone structure has good cell compatibility, the composite chitosan nanoparticles coating is more conducive to the early adhesion growth of osteoblasts. Proliferation and osteogenic differentiation; (3) chitosan coating could effectively inhibit the adhesion of Porphyromonas gingivalis; chitosan nanoparticles had better bacteriostatic effect than chitosan glycerophosphoric acid thermo-sensitive hydrogels.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.1
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