【摘要】：目的 本研究以PGA和β-TCP為基料制備復(fù)合支架材料,并觀察復(fù)合支架材料的微觀結(jié)構(gòu),測試其孔隙率、抗壓強度。將富血小板血漿(Platelet-Rich-Plasma,簡稱PRP)作為內(nèi)源性生長因子與制備出的復(fù)合支架材料復(fù)合研究其對成骨細(xì)胞的增殖、分化作用。為動物實驗及臨床應(yīng)用提供實驗依據(jù)。 方法 1.改良溶液澆鑄-離子瀝濾法制備質(zhì)量比1：1(A組)與1：3(B組)的PGA/β-TCP復(fù)合支架材料,掃描電鏡(SEM)觀察兩組支架材料的微結(jié)構(gòu),用萬能材料試驗機對制備的試樣進行抗壓縮強度的測試,用液體置換法測試PGA/β-TCP復(fù)合支架材料的孔隙率。 2.成骨細(xì)胞的原代培養(yǎng)：無菌條件下處死一日齡SD大鼠,超凈臺下剝離出顱骨片,Ⅰ型膠原酶消化成單個細(xì)胞后以10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)、純化。傳至第三代,用倒置顯微鏡觀察細(xì)胞形態(tài),堿性磷酸酶染色法、茜素紅鈣結(jié)節(jié)染色法鑒定成骨細(xì)胞。 3.取SD大鼠全血,兩步離心法制備PRP,將上步培養(yǎng)的三代成骨細(xì)胞接種于制備好的PGA/β-TCP復(fù)合支架材料上并與PRP復(fù)合。實驗A組為成骨細(xì)胞復(fù)合5%PRP/PGA/β-TCP支架,B組為成骨細(xì)胞復(fù)合10%PRP/PGA/β-TCP支架。對照組為成骨細(xì)胞復(fù)合不加PRP的PGA/β-TCP材料。于培養(yǎng)的1d、2d、3d時做細(xì)胞增殖MTT檢測,2d、4d、6d做堿性磷酸酶定量檢測。 結(jié)果 1.SEM照片可見兩組支架材料均為三維多孔結(jié)構(gòu),孔隙的連通性好。β-TCP均勻地分散于聚合物中,B組支架材料中p-TCP顆粒明顯多于A組。B組支架材料的抗壓縮強度大于A組,差異有統(tǒng)計學(xué)意義(P0.01)；A、B組支架材料孔隙率均大于85%,A組孔隙率大于B組,差異有統(tǒng)計學(xué)意義(P0.01)。 2.倒置顯微鏡下可見48h后細(xì)胞貼壁生長良好,細(xì)胞體積較大,呈三角形、多角形或不規(guī)則形。胞漿豐富并向外伸展,有突起。7d后細(xì)胞長滿培養(yǎng)瓶,細(xì)胞間出現(xiàn)融合,細(xì)胞邊界不清。第三代細(xì)胞堿性磷酸酶染色可見多數(shù)細(xì)胞細(xì)胞核、胞質(zhì)內(nèi)染色呈陽性。鈣結(jié)節(jié)茜素紅染色可見細(xì)胞之間有團塊狀的鈣鹽沉積,呈橘紅色或深紅色。礦化結(jié)節(jié)大小不一,有的多個礦化結(jié)節(jié)可以融合。 3.實驗A、B組與對照組的細(xì)胞增殖隨時間的延長而增加,實驗A、B組較對照組各相同時點細(xì)胞增殖明顯上升,同一時間點,實驗B組測得的OD值最高,差異有統(tǒng)計學(xué)意義(P0.05)；3組在2d時檢測出堿性磷酸酶的活性均較低, 4、6d時三組成骨細(xì)胞ALP的活性隨時間的延長呈增加趨勢,實驗B組成骨細(xì)胞ALP的活性隨時間變化最為明顯,其6d時成骨細(xì)胞ALP含量顯著高于2d時的ALP含量。對照組各相同時點ALP的活性雖也有升高,但都低于實驗兩組。差異有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 1.A、B組PGA/β-TCP復(fù)合支架材料孔隙率均在85%以上,B組復(fù)合支架抗壓縮強度顯著高于A組,B組(1：3質(zhì)量比)PGA/β-TCP復(fù)合支架更能適應(yīng)臨床對支架材料的要求。 2.PRP可以促進PGA/β-TCP復(fù)合支架上成骨細(xì)胞的增殖及提高ALP的活性。其中10%濃度的PRP對PGA/β-TCP組復(fù)合支架材料上成骨細(xì)胞增殖及ALP活性作用要高于5%PRP濃度組。
[Abstract]:objective
In this study, PGA and beta-TCP were used to prepare composite scaffolds, and the microstructure of composite scaffolds was observed, the porosity and compressive strength were tested. It provides experimental evidence for animal experiment and clinical application.
Method
1. The PGA/beta-TCP composite scaffolds with mass ratio of 1:1 (group A) and 1:3 (group B) were prepared by improved solution casting-ion leaching method. The microstructure of the scaffolds was observed by scanning electron microscopy (SEM). The compressive strength of the prepared samples was tested by universal material testing machine. The porosity of PGA/beta-TCP composite scaffolds was measured by liquid displacement method.
2. Primary culture of osteoblasts: one-day-old SD rats were sacrificed under aseptic conditions, skull slices were stripped off under super-clean table, and the collagenase type I was digested into a single cell and cultured in DMEM medium with 10% fetal bovine serum, and then passed to the third generation. Cell morphology was observed by inverted microscope, alkaline phosphatase staining and alizarin red calcium nodule staining. Osteoblasts.
3. PRP was prepared from the whole blood of SD rats by two-step centrifugation method. Three generations of osteoblasts were inoculated into PGA/beta-TCP composite scaffolds and combined with PRP. The osteoblasts in group A were mixed with 5% PRP/PGA/beta-TCP scaffolds, while the osteoblasts in group B were combined with 10% PRP/PGA/beta-TCP scaffolds. Beta -TCP material. MTT was detected in cultured 1D, 2D and 3D, and 2D, 4D and 6D were used for quantitative detection of alkaline phosphatase.
Result
1. SEM images showed that the two groups of scaffolds were three-dimensional porous structure with good pore connectivity. Beta-TCP was dispersed evenly in the polymer. The p-TCP particles in group B were significantly more than those in group A. The compressive strength of group B was higher than that of group A, and the difference was statistically significant (P 0.01). Compared with group B, the difference was statistically significant (P0.01).
2. Under inverted microscope, the cells adhered to the wall and grew well 48 hours later. The cells were large, triangular, polygonal or irregular in size. The cytoplasm was abundant and extended outward with protuberances. After 7 days, the cells grew into culture flasks and fused with each other, and the cell boundary was unclear. Alizarin red staining of calcium nodules showed massive calcium deposits between cells, which were orange or dark red. The mineralized nodules varied in size and some of them could fuse.
3. The proliferation of cells in group A, B and control increased with time. The proliferation of cells in group A and B increased significantly at the same time point compared with the control group. At the same time point, the OD value of group B was the highest, and the difference was statistically significant (P 0.05); the activity of alkaline phosphatase was lower in the three groups at the 2nd day.
The activity of ALP in osteoblasts of experimental group B was the most obvious change with time. The content of ALP in osteoblasts of experimental group B was significantly higher than that of osteoblasts of 2 days at 6 days. The activity of ALP in control group was also increased at the same time, but was lower than that of experimental group (P 0.05). ).
conclusion
1.The porosity of PGA/beta-TCP composite scaffolds in group A and group B was above 85%. The compressive strength of PGA/beta-TCP composite scaffolds in group B was significantly higher than that in group A. The PGA/beta-TCP composite scaffolds in group B (1:3 mass ratio) were more suitable for the clinical requirements of scaffolds.
2. PRP can promote the proliferation of osteoblasts on PGA/beta-TCP composite scaffolds and increase the activity of ALP. The effect of 10% PRP on osteoblasts proliferation and ALP activity on PGA/beta-TCP composite scaffolds was higher than that on 5% PRP group.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08

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