【摘要】：目的 通過(guò)組織工程學(xué)的方法，利用殼聚糖納米微粒的緩釋作用，構(gòu)建rhBMP-2與bFGF序貫緩釋系統(tǒng)，然后復(fù)合于去抗原處理的豬松質(zhì)骨（Antigen-extractedxenogeneic cancellous bone，AXCB），移植于小鼠腓骨肌袋進(jìn)行異體成骨效能研究。通過(guò)動(dòng)物實(shí)驗(yàn)，研究rhBMP-2與bFGF聯(lián)合應(yīng)用時(shí)不同緩釋順序?qū)θタ乖i松質(zhì)骨異體移植的成骨效能的影響，探討rhBMP-2與bFGF分別在骨形成過(guò)程中的作用機(jī)制及相互作用的機(jī)制。設(shè)計(jì)并證明具有良好促骨形成效能的rhBMP-2與bFGF序貫緩釋系統(tǒng)，為進(jìn)一步的實(shí)驗(yàn)研究及以后的臨床應(yīng)用提供理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。 方法 實(shí)驗(yàn)一：采用表面涂覆/沉積的方法，利用殼聚糖納米顆粒包裹rhBMP-2小分子與bFGF小分子構(gòu)建CS/rhBMP-2/bFGF序貫緩釋系統(tǒng)，并將該復(fù)合納米顆粒涂覆于去抗原處理后的豬松質(zhì)骨塊的骨小梁結(jié)構(gòu)表面，然后掃描電鏡觀察形態(tài)、比重法檢測(cè)孔隙率，壓縮模量測(cè)試檢測(cè)生物力學(xué)性能； 實(shí)驗(yàn)二：進(jìn)行材料載藥與體外釋放實(shí)驗(yàn)，通過(guò)包封率和載藥量的測(cè)定來(lái)確定材料的載藥能力，并計(jì)算藥物的緩釋時(shí)間，描繪緩釋曲線，驗(yàn)證復(fù)合移植骨材料的成功制備。 實(shí)驗(yàn)三：設(shè)計(jì)動(dòng)物實(shí)驗(yàn)研究方案，進(jìn)行材料組織相容性及異體成骨效能的研究。選取約30g重的昆明SD小鼠64只，隨機(jī)分為實(shí)驗(yàn)組及對(duì)照組兩大組，其中實(shí)驗(yàn)組按照CS/rhBMP-2/bFGF序貫緩釋順序的不同分為6組：①實(shí)驗(yàn)組A：AXCB+rhBMP-2/bFGF，②實(shí)驗(yàn)組B：AXCB+CS/rhBMP-2/bFGF，③實(shí)驗(yàn)組C：AXCB+rhBMP-2/CS/bFGF，④實(shí)驗(yàn)組D：AXCB+bFGF/CS/rhBMP-2，⑤實(shí)驗(yàn)組E：AXCB+CS/rhBMP-2，⑥實(shí)驗(yàn)組F：AXCB+CS/bFGF；對(duì)照組又分為兩組：⑦空白對(duì)照組G：AXCB+CS，及⑧陰性對(duì)照組H：不植入任何材料組。于2w、4w兩個(gè)時(shí)間點(diǎn)分別處死動(dòng)物后獲取標(biāo)本，進(jìn)行大體標(biāo)本觀察與稱(chēng)重、μCT觀察骨組織顯微三維結(jié)構(gòu)、骨參數(shù)測(cè)量分析、鈣含量檢測(cè)、組織形態(tài)學(xué)染色（HE染色）以及組織化學(xué)染色（ALP免疫組化染色及CD34免疫組化染色）等。對(duì)實(shí)驗(yàn)得到的所有數(shù)據(jù)均以x±s表示，應(yīng)用SPSS13.0軟件，采用完全隨機(jī)設(shè)計(jì)的單因素方差分析、多個(gè)樣本均數(shù)兩兩比較的SNK-q檢驗(yàn)等方法對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析，以α=0.05為檢驗(yàn)水準(zhǔn)，規(guī)定P0.05為差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果 實(shí)驗(yàn)一：AXCB/CS/rhBMP/bFGF序貫緩釋系統(tǒng)的制備與表征 1.1掃描電鏡結(jié)果顯示去抗原豬松質(zhì)骨塊具有疏松多孔狀結(jié)構(gòu)，且附載的CS/rhBMP-2/bFGF緩釋系統(tǒng)呈約100納米的顆粒狀結(jié)構(gòu)吸附于骨小梁表面； 1.2孔隙率測(cè)定結(jié)果顯示去抗原豬松質(zhì)骨塊具有高達(dá)85%的孔隙率，且吸附不同緩釋順序的生長(zhǎng)因子納米顆粒后，其孔隙率均沒(méi)有發(fā)生較大的改變，均保持在85%-86%之間； 1.3力學(xué)性能分析結(jié)果顯示去抗原豬松質(zhì)骨塊的壓縮模量為13.44MPa，吸附不同緩釋順序的生長(zhǎng)因子納米顆粒后，其壓縮模量均沒(méi)有發(fā)生較大的改變，均保持在12MPa-14MPa之間，且其強(qiáng)度均能達(dá)到骨組織工程對(duì)于植入材料強(qiáng)度的要求； 實(shí)驗(yàn)二：AXCB/CS/rhBMP/bFGF序貫緩釋系統(tǒng)的載藥及釋放研究 2.1各組材料中rhBMP-2及bFGF的包封率均較為接近，分別在55%及60%以上；且各組材料中rhBMP-2及bFGF的載藥量也較為接近，分別在22‰及0.012‰左右； 2.2體外釋放試驗(yàn)所描繪的釋放曲線可知，rhBMP-2與bFGF同時(shí)暴釋組分別在第9天達(dá)到rhBMP80%的累積釋放率及第13天達(dá)到bFGF70%的累積釋放率；rhBMP-2與bFGF同時(shí)緩釋組則rhBMP-2與bFGF持續(xù)釋放至第23天；rhBMP-2先暴釋?zhuān)琤FGF后緩釋組中rhBMP-2第9天就達(dá)到80%的累積釋放率，而bFGF第8天開(kāi)始釋放，持續(xù)釋放至第26天累積達(dá)70%；bFGF先暴釋?zhuān)瑀hBMP-2后緩釋組中bFGF第13天就達(dá)到70%的累積釋放率，rhBMP-2第7天開(kāi)始釋放，持續(xù)釋放至第28天累積達(dá)80%。單純r(jià)hBMP-2緩釋組中rhBMP-2持續(xù)釋放至第24天時(shí)累積釋放率才達(dá)到80%；單純bFGF緩釋組中bFGF持續(xù)釋放至第22天時(shí)累積釋放率才達(dá)到70%。 實(shí)驗(yàn)三：AXCB/CS/rhBMP/bFGF序貫緩釋系統(tǒng)的異體成骨實(shí)驗(yàn) 3.1大體觀察及稱(chēng)重結(jié)果顯示，A-2、B-2、C-2、A-4、B-4、C-4、D-4、E-4材料重量明顯高于術(shù)前材料重量（P0.05）；2周時(shí)A組材料重量高于其他組別（P0.05）；4周時(shí)B組材料重量高于其他組別（P0.05）；且B、E組4周時(shí)材料重量高于2周時(shí)（P0.05）； 3.2鈣含量檢測(cè)結(jié)果顯示2周時(shí)各組材料鈣含量濃度值比較為A、C組B組D、E、F、G組(P0.05)，且A組的鈣含量濃度最大(348.5±9.66mg/dL)；4周時(shí)為B組A、C、D、E組F、G組(P0.05)，且B組的鈣含量濃度最大(414.7±12.03mg/dL)。同時(shí)B、D、E組4周時(shí)的鈣含量濃度均高于2周時(shí)(P0.05)，分別高出123.1mg/dL、78.2mg/dL、88.3mg/dL; 3.3μCT掃描及骨參數(shù)分析結(jié)果顯示，所有植入的去抗原豬松質(zhì)骨塊的骨體積、骨表面積及骨體積分?jǐn)?shù)均相近(P0.05)；而骨礦物質(zhì)密度分析發(fā)現(xiàn)，2周時(shí)各組材料骨密度值比較為A、C、E組B組D、F、G高(P0.05)，且A組的骨密度值最大(307.09±8.27mg/cc)；4周時(shí)為B組A、C、D、E組F、G組(P0.05)，且B組的骨密度值最大(367.52±11.64mg/cc)；同時(shí)A、B、D、E組4周時(shí)的骨密度值均高于2周時(shí)(P0.05)，分別高出32.55mg/cc、89.06mg/cc、38.18mg/cc、29.57mg/cc；骨小梁厚度分析發(fā)現(xiàn)，2周時(shí)各組材料骨小梁厚度比較為A組B、C、E組D、F、G高(P0.05)，且A組的骨小梁厚度最大(95.33±7.28μm)；4周時(shí)為B組A、C、D組E、F、G組(P0.05)，且B組的骨小梁厚度最大(126.17±11.36μm)；同時(shí)A、B、C、D組4周時(shí)的骨小梁厚度均高于2周時(shí)(P0.05)，分別高出14.51μm、39.75μm、23.15μm、22.49μm； 3.4H.E.染色結(jié)果顯示A組、B組、C組、D組及E組在組織學(xué)HE染色結(jié)果中均可見(jiàn)較大區(qū)域的鈣化軟骨及造血組織的形成，在新生的鈣化軟骨上可見(jiàn)透亮的軟骨細(xì)胞及軟骨邊緣可見(jiàn)的骨母細(xì)胞襯里于軟骨邊緣，同時(shí)，在新生的軟骨與植入的異種骨小梁及周?chē)浗M織之間可見(jiàn)紅細(xì)胞充盈的造血組織，其中深染的細(xì)胞為造血細(xì)胞，紅染的細(xì)胞為血紅細(xì)胞。 3.5堿性磷酸酶免疫組化染色結(jié)果顯示，全部植入材料的小鼠部位均可見(jiàn)不同程度程度的ALP陽(yáng)性表達(dá)區(qū)域，主要位于新生軟骨的外周，表達(dá)于軟骨細(xì)胞的胞漿內(nèi)。其中A-2組、A-4組、B-4組、C-4組、D-4組及E-4組中ALP的陽(yáng)性表達(dá)要高于其他組； 3.6CD34免疫組化染色結(jié)果顯示全部植入材料的小鼠部位均可見(jiàn)不同程度程度的CD34陽(yáng)性表達(dá)區(qū)域，主要表達(dá)于血管內(nèi)皮細(xì)胞的胞膜和胞漿。A-2組、A-4組、B-4組、C-4組、D-4組及F-4組中CD34陽(yáng)性表達(dá)要高于其他組。 結(jié)論 1本實(shí)驗(yàn)已成功完成對(duì)AXCB/CS/rhBMP-2/bFGF序貫緩釋系統(tǒng)的制備、表征、體外釋放及小鼠體內(nèi)成骨效能的研究。 2本實(shí)驗(yàn)制備的AXCB/CS/rhBMP-2/bFGF序貫緩釋系統(tǒng)具有理想的疏松多孔狀結(jié)構(gòu)，，生物力學(xué)性能滿(mǎn)足骨組織工程要求，顯微結(jié)構(gòu)形貌觀察可見(jiàn)殼聚糖包裹生長(zhǎng)因子的納米顆粒成功地吸附于骨小梁結(jié)構(gòu)表面，且各組材料中rhBMP-2與bFGF的總量及其各自所占移植材料總質(zhì)量的比例之間無(wú)差異； 3體外釋放實(shí)驗(yàn)證實(shí)了AXCB/CS/rhBMP-2/bFGF序貫緩釋系統(tǒng)的成功制備，各組材料中rhBMP-2與bFGF均按照實(shí)驗(yàn)設(shè)計(jì)實(shí)現(xiàn)先后的緩慢釋放順序。 4動(dòng)物實(shí)驗(yàn)結(jié)果均表明2周時(shí)，rhBMP-2與bFGF同時(shí)暴釋組誘導(dǎo)去抗原豬松質(zhì)骨的成骨效能最高；而4周時(shí)，則是rhBMP-2與bFGF同時(shí)緩釋組誘導(dǎo)去抗原豬松質(zhì)骨的成骨效能最高；鑒于新骨形成及礦化的周期時(shí)間，rhBMP-2與bFGF同時(shí)緩釋是最為理想的釋藥方式，對(duì)去抗原豬松質(zhì)骨異體移植具有更好的誘導(dǎo)成骨作用。
[Abstract]:objective
By means of tissue engineering, the slow release of chitosan nanoparticles was used to construct rhBMP-2 and bFGF sequential sustained release system, and then combined with Antigen-extractedxenogeneic cancellous bone (AXCB), which was treated with antigen treatment, and transplanted into the fibula muscle bag of mice to study the osteogenic effect of allogenic bone. The effect of different slow release sequence on the osteogenesis of allogenic porcine cancellous bone allograft in combination with bFGF, and the mechanism of action and interaction between rhBMP-2 and bFGF during bone formation, and the design and demonstration of rhBMP-2 and bFGF sequential sustained release system with good bone formation efficiency for further experimental study and It provides theoretical basis and experimental evidence for future clinical application.
Method
Experiment 1: using the method of surface coating / deposition, chitosan nanoparticles were used to encapsulate rhBMP-2 small molecules and bFGF small molecules to construct CS/rhBMP-2/bFGF sequential slow release system, and the composite nanoparticles were coated on the surface of bone trabecular structure of porcine cancellous bone mass after antigenic treatment, and then scanning electron microscopy was used to observe morphology and specific gravity method to detect holes. The porosity and compression modulus were tested to detect biomechanical properties.
Experiment two: the drug loading and in vitro release experiment were carried out. The drug carrying capacity of the material was determined by the encapsulation rate and the measurement of drug loading, and the release time of the drug was calculated, the sustained release curve was depicted, and the successful preparation of the composite bone graft was verified.
Experiment three: Design animal experimental research program, study the tissue compatibility and allogenic osteogenesis efficiency of materials, select 64 Kunming SD mice with about 30g weight, randomly divide into experimental group and control group two groups, and the experimental group is divided into 6 groups according to the sequence of CS/rhBMP-2/bFGF sequential slow release: (1) the experimental group A:AXCB+rhBMP-2/bFGF, 2 The test group B:AXCB+CS/rhBMP-2/bFGF, the experimental group C:AXCB+rhBMP-2/CS/bFGF, the experimental group D:AXCB+bFGF/CS/rhBMP-2, the experimental group E:AXCB+CS/rhBMP-2, and the experimental group F:AXCB+CS/bFGF; the control group is divided into two groups: the blank control group G:AXCB+CS, and the negative control group H: not in any material group. In 2W, 4W two time points. After the animals were executed, the specimens were collected and the specimen was observed and weighed. The microstructure of the bone tissue was observed by micron CT, the bone parameters were measured and analyzed, the calcium content was detected, the histomorphological staining (HE staining) and histochemical staining (ALP immunohistochemical staining and CD34 immunohistochemical staining) were used. All the data obtained by the experiment were x + s, SPSS13.0 software is used to analyze the data by means of a single factor analysis of variance and SNK-q test of multiple samples, which are compared with two or two, with a test level of alpha =0.05, and the difference between P0.05 and P0.05 has statistical significance.
Result
Experiment 1: preparation and characterization of AXCB/CS/rhBMP/bFGF sequential release system
1.1 the results of scanning electron microscopy showed that the antigenic porcine cancellous bone block had porous and porous structure, and the loaded CS/rhBMP-2/bFGF sustained release system was about 100 nanometers of granular structure adsorbed on the surface of bone trabecula.
The results of 1.2 porosity determination showed that the porcine cancellous bone mass of the antiantigen was up to 85% porosity, and the porosity of the growth factor nanoparticles had not changed greatly after the adsorption of different slow release order growth factor nanoparticles, and all remained at 85%-86%.
1.3 the results of mechanical properties analysis showed that the compression modulus of the antigenic porcine cancellous bone block was 13.44MPa, and the compression modulus was not changed greatly after the adsorption of different slow release growth factor nanoscale nanoparticles, and the strength of the bone fabric could meet the requirement of the strength of the implant.
Experiment two: drug loading and release of AXCB/CS/rhBMP/bFGF sequential release system
2.1 the encapsulation efficiency of rhBMP-2 and bFGF in all kinds of materials were all close to 55% and 60%, respectively, and the drug loading of rhBMP-2 and bFGF in each group were also close, respectively, at 22 per thousand and 0.012 per thousand, respectively.
The release curve depicted in 2.2 in vitro release test showed that the cumulative release rate of rhBMP-2 and bFGF reached rhBMP80% at ninth days and the cumulative release rate of bFGF70% on the 13 day, while rhBMP-2 and bFGF sustained release to twenty-third days in rhBMP-2 and bFGF simultaneous release group, rhBMP-2 first release and rhBMP-2 ninth days in bFGF after release group. The cumulative release rate of 80% was reached, while bFGF eighth days began to release, sustained release to 70% in twenty-sixth days; bFGF was first released, and the cumulative release rate of bFGF thirteenth days in rhBMP-2 release group was reached, rhBMP-2 seventh days began to release, and continued to release to twenty-eighth days to accumulate to accumulate to twenty-fourth days when rhBMP-2 continued to accumulate to twenty-fourth days. The release rate reached 80%, and the sustained release rate of bFGF in the bFGF slow release group reached 70%. only after twenty-second days.
Experiment three: allogeneic osteogenesis of AXCB/CS/rhBMP/bFGF sequential release system.
3.1 gross observation and weighing results showed that the weight of A-2, B-2, C-2, A-4, B-4, C-4, D-4, E-4 was significantly higher than the weight of pre operation material (P0.05), and at the 2 week, the weight of the A group was higher than that of the other groups (P0.05), and the material weight of the B group was higher than that of the other groups at 4 weeks, and the weight of the material was higher than 2 weeks at the week of 4.
The results of 3.2 calcium content test showed that the calcium content of each group was A at 2 weeks, C group B group D, E, F, G group (P0.05), and the concentration of calcium content in A group was maximum (348.5 + 9.66mg/dL), and 4 weeks was B group A, and the concentration of calcium content was the largest (414.7 +). The time (P0.05) is higher than 123.1mg/dL, 78.2mg/dL and 88.3mg/dL respectively.
3.3 CT scanning and bone parameter analysis showed that the bone volume, bone surface area and bone volume fraction of all implanted antigen porcine cancellous bone were similar (P0.05), while bone mineral density analysis showed that the bone mineral density of each group was A, C, B group D, F, G high (P0.05) at the time of 2 weeks, and the BMD value was maximum (307.09 + 8.27mg/cc) in A group (307.09 + 8.27mg/cc); 4 B group A, C, D, E group F, G group (P0.05), and B group (P0.05) in B group (367.52 + 11.64mg/cc). (P0.05), and the thickness of bone trabecula in group A was maximum (95.33 + 7.28 m), and at 4 weeks, B group A, C, D group E, F, G group (P0.05), and B group bone trabecular thickness was maximum (126.17 + 11.36 micron). At the same time, the small bone Liang Houdu of 4 weeks was higher than 2 weeks, 39.75 mu, 23.15 mu, 22.49 micron;
The results of 3.4H.E. staining showed that in group A, group B, group C, group D and group E, the formation of calcified cartilage and hematopoietic tissue in the larger region were found in the histological HE staining results, and the bright chondrocytes and cartilage margins were visible on the edge of cartilage on the newborn calcified cartilage, while the new cartilage and the implanted xenogeneic bone were found. Red blood cells filled with hematopoietic tissue were found between trabecular and surrounding soft tissues. Deep stained cells were hematopoietic cells and red stained cells were erythrocytes.
3.5 the results of immuno histochemical staining of alkaline phosphatase showed that all the implanted materials showed ALP positive expression regions of different degrees, mainly located in the outer circumference of the newborn cartilage, and expressed in the cytoplasm of chondrocytes. The positive expression of ALP in group A-2, group A-4, B-4, C-4, D-4 and E-4 groups was higher than that of other groups.
The results of 3.6CD34 immunohistochemical staining showed that all the implanted materials showed different degrees of CD34 positive expression in different degrees, mainly expressed in the cell membrane and cytoplasm.A-2 group of vascular endothelial cells, and the positive expression of CD34 in group A-4, B-4 group, C-4 group, D-4 group and F-4 group was higher than that of other groups.
conclusion
1 this experiment has successfully completed the preparation, characterization, in vitro release and osteogenic efficiency of AXCB/CS/rhBMP-2/bFGF sequential release system.
2 the AXCB/CS/rhBMP-2/bFGF sequential sustained release system prepared by this experiment has ideal loose porous structure and the biomechanical properties meet the requirements of bone tissue engineering. The microstructure and morphology observe that the chitosan coated growth factor nanoparticles have been successfully adsorbed on the surface of the bone trabecular structure, and the total amount of rhBMP-2 and bFGF in each material and the total amount of the particles in the materials are also observed. There was no difference in the proportion of total mass of transplanted materials.
3 in vitro release test confirmed the successful preparation of AXCB/CS/rhBMP-2/bFGF sequential sustained release system. Both rhBMP-2 and bFGF in all the materials were designed to achieve the slow release sequence according to the experimental design.
The results of 4 animal experiments showed that at 2 weeks, the osteogenic efficacy of rhBMP-2 and bFGF induced antigenic porcine cancellous bone was highest, while at 4 weeks, it was the highest osteogenic efficacy of rhBMP-2 and bFGF simultaneous release group to induce antigenic porcine cancellous bone. In view of the period of new bone formation and mineralization, the simultaneous release of rhBMP-2 and bFGF was the most ideal. The release method has a better osteogenic effect on xenotransplantation of cancellous bone.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R782.2

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