本文選題：PFTBA + GelMA水凝膠��； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：嚴(yán)重骨折和骨缺損導(dǎo)致骨的連續(xù)性和結(jié)構(gòu)完整性被破壞,導(dǎo)致患肢功能受損甚至喪失功能,嚴(yán)重影響著患者的生活質(zhì)量,是骨科領(lǐng)域急需解決的難題之一。骨內(nèi)血管分布較為豐富,為骨組織正常的功能運(yùn)轉(zhuǎn)提供相應(yīng)的氧氣和營養(yǎng)成分。大量研究表明:骨折后,骨折處血管遭到大量破壞,使受損處組織的氧份和營養(yǎng)供應(yīng)不足,嚴(yán)重影響骨折后的修復(fù)。如果缺損過大(≥8 mm),其自身很難完全愈合,易造成骨延遲愈合或骨不連等并發(fā)癥的發(fā)生。組織工程和生物材料技術(shù)的應(yīng)用為骨修復(fù)提供了新的方法。但是,應(yīng)當(dāng)注意到在缺氧條件下,種子細(xì)胞的存活和相應(yīng)的功能發(fā)揮受到極大的抑制,這影響了其修復(fù)骨缺損的效能。因此,嚴(yán)重骨折和骨缺損后,缺損區(qū)域的氧氣供應(yīng)對(duì)骨修復(fù)十分重要�，F(xiàn)如今有很多攜氧和釋放氧氣的材料被用于缺氧的研究。全氟三丁胺(Perfluorotributylamine,PFTBA)因其具有較好的氧溶解性和無毒副作用,被廣泛用于解決組織缺氧的研究中。但是,PFTBA釋放氧氣的時(shí)間為4-5天,因此如果將PFTBA應(yīng)用于骨缺損的修復(fù),還需考慮4-5天后的氧氣供應(yīng)。新生血管可以為骨缺損處提供氧氣,血管組織分為三層,分別為內(nèi)膜、中膜和外膜,其中最里層的內(nèi)皮細(xì)胞對(duì)新生血管的形成起著關(guān)鍵的作用。如果將此細(xì)胞應(yīng)用于骨缺損的修復(fù),可以加速缺損處的血管化進(jìn)程。因此為更好地促進(jìn)骨折或骨缺損后的骨修復(fù),本研究嘗試制做一種復(fù)合有攜氧材料PFTBA的甲基丙烯酸酯明膠Gelatin methacryloyl(Gel MA)支架材料,并將脂肪干細(xì)胞與血管內(nèi)皮細(xì)胞復(fù)合進(jìn)此材料,用于嚴(yán)重骨折或者骨缺損修復(fù)的相關(guān)研究,以期在骨缺損的修復(fù)過程中為其提供充足的氧氣且加速骨折處的血管再生,促進(jìn)受損區(qū)骨組織更快更好的修復(fù)。整個(gè)研究分為以下三個(gè)部分:實(shí)驗(yàn)一:PFTBA-Gel MA水凝膠支架的制備及相關(guān)理化性質(zhì)和生物性能檢測(cè)背景:嚴(yán)重骨折和骨缺損導(dǎo)致患肢功能受限甚至喪失功能,嚴(yán)重影響著患者的生活質(zhì)量。自體或異體骨移植是治療骨缺損的最有效的辦法,但是由于骨的供源不足,取骨過程易造成二次損傷,以及異體骨產(chǎn)生的免疫排斥反應(yīng),均限制了其應(yīng)用。組織工程技術(shù)的發(fā)展為解決這一難題提供了新的方法。載體生物支架作為組織工程的三要素之一,應(yīng)具有良好的生物相容性和可降解性,能夠促進(jìn)細(xì)胞的存活,增殖,分化,黏附,另外有研究指出不同機(jī)械強(qiáng)度的細(xì)胞微環(huán)境能促進(jìn)細(xì)胞產(chǎn)生不同的功能應(yīng)答,因此該生物支架應(yīng)該具有可調(diào)節(jié)的機(jī)械力學(xué)性能。同時(shí),在體內(nèi),細(xì)胞均立體生活在相應(yīng)的生物基質(zhì)內(nèi)。模擬細(xì)胞的生存環(huán)境對(duì)細(xì)胞功能的發(fā)揮十分有利,現(xiàn)如今大量水凝膠材料應(yīng)用于細(xì)胞的三維培養(yǎng),包括膠原蛋白(Collagen),藻酸鹽(Alginate),透明質(zhì)酸(Hyaluronic acid,HA),Gel MA等。其中,Gel MA水凝膠材料以其易合成,造價(jià)低,生物相容性較好,可調(diào)節(jié)機(jī)械強(qiáng)度等優(yōu)點(diǎn)被廣泛應(yīng)用于組織工程領(lǐng)域。骨缺損造成的低氧環(huán)境不利于骨組織的修復(fù)。PFTBA是一種很好的攜氧材料,在本實(shí)驗(yàn)中,我們將其與Gel MA混合,制作出PFTBA-Gel MA水凝膠,以期在應(yīng)用中釋放足夠的氧氣為骨修復(fù)所用。目的:構(gòu)建生物相容性較好的PFTBA-Gel MA水凝膠材料,并檢測(cè)其理化性質(zhì)和生物學(xué)性能。方法:采用化學(xué)合成的方法制作出Gel MA水凝膠,將不同濃度的PFTBA(0,5%,10%)整合進(jìn)Gel MA水凝膠內(nèi),采用不同的紫外光照時(shí)間交聯(lián),制作出不同硬度的水凝膠材料。用Instron機(jī)器來檢測(cè)各組水凝膠的楊氏模量。以此水凝膠為3D培養(yǎng)支架,復(fù)合進(jìn)ADSCs,缺氧環(huán)境下(1%O2)觀察該P(yáng)FTBA-Gel MA水凝膠對(duì)ADSCs細(xì)胞活性的影響(Presto Blue實(shí)驗(yàn))。Live/Dead實(shí)驗(yàn)檢測(cè)細(xì)胞在該水凝膠內(nèi)的存活狀態(tài),統(tǒng)計(jì)其存活細(xì)胞率。結(jié)果:當(dāng)紫外光照時(shí)間分別為15,20,25,30 s時(shí),各組水凝膠的強(qiáng)度分別約為13.87±1.30 Kpa,19.76±1.61Kpa,25.43±1.89Kpa,31.23±3.21Kpa。Presto Blue結(jié)果表明,10%PFTBA組能夠很好的促進(jìn)缺氧組細(xì)胞的增殖。Live/Dead熒光染色顯示:相比對(duì)照組,10%PFTBA組和正常氧含量組存活的細(xì)胞較多,且細(xì)胞在PFTBA-Gel MA水凝膠里伸展較好。結(jié)論:通過本實(shí)驗(yàn)方法制作的PFTBA-Gel MA水凝膠能夠根據(jù)紫外光照時(shí)間來調(diào)節(jié)水凝膠的機(jī)械強(qiáng)度;該水凝膠具有良好的生物相容性,能為缺氧環(huán)境下的細(xì)胞提供足夠的氧氣,使其保持較好的細(xì)胞活性。實(shí)驗(yàn)二:缺氧條件下,PFTBA-GelMA水凝膠對(duì)ADSCs成骨分化和VECs血管化的影響背景:骨折或骨缺損發(fā)生后,受損區(qū)域血管遭到大量破壞,無法為受損區(qū)域輸送足夠的氧氣,有研究指出氧分壓在正常骨的含量為~12.5%,當(dāng)骨折或骨缺損發(fā)生時(shí),氧含量迅速降到~1%,造成大量細(xì)胞的處于缺氧環(huán)境,引起細(xì)胞壞死,嚴(yán)重影響了骨缺損后的修復(fù)。且不同氧分壓也嚴(yán)重影響著細(xì)胞的功能發(fā)揮。據(jù)報(bào)道,當(dāng)組織工程支架長(zhǎng)期處于低氧條件下時(shí),會(huì)造成支架中心大量的細(xì)胞壞死。可見,骨折后的氧供應(yīng)不僅影響著骨折本身的再生,還影響著組織工程支架功能的發(fā)揮。因此,骨缺損后的氧供應(yīng)十分必要。在本實(shí)驗(yàn)中我們使用一種能夠釋放氧氣的材料PFTBA-Gel MA水凝膠,再復(fù)合以ADSCs和VECs,觀察其對(duì)它們成骨和血管化的影響。方法:將不同濃度的PFTBA(0%,5%,10%)整合進(jìn)Gel MA水凝膠內(nèi),以此體系為3D培養(yǎng)支架,分別復(fù)合進(jìn)ADSCs,VECs,并將其放置缺氧環(huán)境(1%O2)下,觀察PFTBA對(duì)ADSCs成骨分化,VECs血管化的影響;通過ALP,茜素紅染色和免疫熒光染色,檢測(cè)ADSCs的早期成骨分化,和晚期鈣結(jié)節(jié)的形成情況。對(duì)VECs成血管情況進(jìn)行觀察,并對(duì)總血管形成長(zhǎng)度和血管數(shù)量進(jìn)行統(tǒng)計(jì),研究不同組細(xì)胞的血管化情況。對(duì)ADSCs組和VECs組分別進(jìn)行相關(guān)基因檢測(cè)(成骨相關(guān)基因:ALP,BMP-2,RUNX2,OPN;成血管相關(guān)基因:CD31,VEGF)。將ADSCs單層或者和VECs按1:1比例進(jìn)行共培養(yǎng),分別將其置于缺氧環(huán)境下,對(duì)其進(jìn)行相關(guān)基因檢測(cè),觀察PFTBA對(duì)單層細(xì)胞,和ADSCs-VECs共培養(yǎng)體系的作用。結(jié)果:ALP成骨染色和茜素紅染色結(jié)果顯示10%PFTBA組的細(xì)胞成骨分化效果較好,OPN/F-actin熒光雙標(biāo)染色也得到了類似的結(jié)果。通過對(duì)VECs成血管進(jìn)行研究,發(fā)現(xiàn)12 h后10%PFTBA組成血管的能力最好。基因檢測(cè)表明在10%PFTBA組,成骨相關(guān)基因ALP,RUNX2,OPN,BMP-2的表達(dá)和正常氧含量組無差異,和單層缺氧組和5%PFTBA組相比顯著提高。在共培養(yǎng)體系下,基因檢測(cè)表明10%PFTBA條件下,相比單層培養(yǎng)組,共培養(yǎng)組能更好的促進(jìn)成骨分化和血管化。結(jié)論:缺氧條件下,10%PFTBA-GelMA水凝膠能顯著促進(jìn)ADSCs的成骨分化和VECs的成血管。此作用在共培養(yǎng)條件下得到進(jìn)一步加強(qiáng)。實(shí)驗(yàn)三:復(fù)合ADSCs和VECs的PFTBA-Gel MA水凝膠支架對(duì)骨修復(fù)的體內(nèi)研究背景:創(chuàng)傷、感染、腫瘤、骨髓炎手術(shù)清創(chuàng)等易導(dǎo)致骨缺損的發(fā)生,自體骨移植,異體骨移植,生物陶瓷等應(yīng)用為骨缺損的修復(fù)提供了可能。但是,同時(shí)應(yīng)注意到骨來源有限,免疫排斥反應(yīng),生物相容性等因素限制了這些骨材料的應(yīng)用。組織工程的發(fā)展為骨缺損的修復(fù)提供了新的思路。但是,骨缺損發(fā)生后,缺損區(qū)域遭到破壞的血管無法為組織再生支架提供足夠的氧氣,且缺氧條件下還限制骨本身的自我修復(fù)。因此,解決缺損區(qū)域的氧供應(yīng)成為了目前一項(xiàng)亟需解決的問題。本研究采用的復(fù)合ADSCs和VECs的PFTBA-Gel MA水凝膠已被證明能夠很好地在體外促進(jìn)成骨分化和血管化,但是在體內(nèi)是否有效還未得到證實(shí)。目的:研究復(fù)合ADSCs和VECs的PFTBA-Gel MA水凝膠對(duì)體內(nèi)異位成骨和骨缺損的修復(fù)作用。方法:實(shí)驗(yàn)分組:Control組,ADSCs+VECs組,Cells+PFTBA組。將各組水凝膠分別移植入裸鼠背部皮下。2周,4周取材進(jìn)行HE染色,觀察異位成骨情況。將各組水凝膠移植入大鼠顱骨直徑為5mm的骨缺損。分別在2,4周取材,進(jìn)行Micro-CT掃描檢測(cè),檢測(cè)其成骨能力。對(duì)實(shí)驗(yàn)樣本進(jìn)行基因檢測(cè),檢測(cè)其成骨和成血管相關(guān)基因的表達(dá)情況。結(jié)果:HE染色結(jié)果顯示Cells+PFTBA組有大量骨基質(zhì)形成,相比Control組和ADSCs+VECs組,骨基質(zhì)的形成量顯著增加。Micro-CT結(jié)果表明,4周后,與Control組和ADSCs+VECs組相比,Cells+PFTBA組的新生骨量顯著增多。基因檢測(cè)表明,與Control組相比,Cells+PFTBA組成骨和血管化相關(guān)基因的表達(dá)均顯著上升。結(jié)論:復(fù)合ADSCs和VECs細(xì)胞的PFTBA-Gel MA水凝膠支架能夠促進(jìn)異位成骨,并能較快的修復(fù)大鼠顱骨骨缺損。
[Abstract]:Severe fracture and bone defect cause bone continuity and structural integrity to be destroyed, resulting in damaged limb function and even loss of function. It seriously affects the quality of life of the patients. It is one of the difficult problems to be solved in the field of Department of orthopedics. The distribution of internal blood vessels is abundant, which provides the corresponding oxygen and nutrition components for the normal function of bone tissue. A large number of studies have shown that after fracture, the blood vessels of the fracture are badly damaged and the oxygen and nutrition supply of the damaged tissue is insufficient and the repair of the fracture is seriously affected. If the defect is too large (> 8 mm), it is difficult to complete the complete healing of the bone. It is easy to cause the complications such as delayed union or nonunion of bone. It is a new method for bone repair. However, it should be noted that under the condition of hypoxia, the survival of the seed cells and the corresponding function are greatly suppressed, which affects its effectiveness in repairing bone defects. Therefore, oxygen supply in the defect area is very important for bone repair after severe fracture and bone defect. Now, there are many oxygen carrying and release. Oxygen - releasing materials are used for anoxia. Perfluorotributylamine (Perfluorotributylamine, PFTBA) is widely used in the study of tissue anoxia because of its good oxygen solubility and non-toxic side effects. However, the time for PFTBA release of oxygen is 4-5 days. Therefore, if PFTBA is applied to bone defect repair, it should be considered for 4-5 days. Oxygen supply. Neovascularization can provide oxygen for bone defect. Vascular tissue is divided into three layers: intima, middle membrane and outer membrane. The most inner layer of endothelial cells plays a key role in the formation of neovascularization. If this cell is used to repair bone defects, it can accelerate the vascularization process at the defect. To promote bone repair after fracture or bone defect, this study attempts to make a composite of methacrylate gelatin Gelatin methacryloyl (Gel MA) scaffold with a compound oxygen carrying material PFTBA, and combine fat stem cells with vascular endothelial cells into this material for severe fracture or bone defect repair in order to be in bone defect. The whole study is divided into three parts: the preparation of PFTBA-Gel MA hydrogel scaffold and the related physical and chemical properties and biological properties detection background: severe fracture and bone defect cause the function of the affected limb. Limit or even loss of function seriously affects the quality of life of the patient. Autologous or allograft bone graft is the most effective method for the treatment of bone defects. However, due to the lack of bone supply, the process of bone taking is easy to cause two damage and the immune rejection of allograft, which restricts its application. The development of tissue engineering technology to solve this difficult problem. It provides a new method. As one of the three elements of tissue engineering, the carrier biological scaffold should have good biocompatibility and biodegradability, which can promote cell survival, proliferation, differentiation and adhesion. In addition, it is pointed out that the cell microenvironment with different mechanical strength can promote the cell to produce different functional responses, so the biological scaffold It should have adjustable mechanical properties. At the same time, the cells live in the corresponding biological matrix in the body. The survival environment of the cells is very beneficial to the function of cells. Nowadays, a large number of hydrogel materials are used in the three-dimensional culture of cells, including collagen protein (Collagen), alginate (Alginate), hyaluronic acid (Hya) Luronic acid, HA), Gel MA and so on. Among them, Gel MA hydrogel material is widely used in the field of tissue engineering for its advantages of easy synthesis, low cost, good biocompatibility and adjustable mechanical strength. The hypoxia environment caused by bone defect is not conducive to bone tissue repair.PFTBA as a good oxygen carrying material. In this experiment, we and Gel in this experiment, we and Gel MA mixed and produced PFTBA-Gel MA hydrogel in order to release sufficient oxygen for bone repair in application. Objective: to construct a biocompatible PFTBA-Gel MA hydrogel material and to detect its physicochemical properties and biological properties. Method: Gel MA hydrogel was made by chemical synthesis, and PFTBA (0,5%, 10%) of different concentrations was obtained. In the Gel MA hydrogel, the hydrogels with different hardness were made by cross-linking with different ultraviolet light time. The young's modulus of each hydrogel was detected by Instron machine. The hydrogel was used as the 3D scaffold, ADSCs and 1%O2 were used to observe the effect of the PFTBA-Gel MA hydrogel on the activity of ADSCs cells (Presto). Blue experiment) the survival state of the cells in the hydrogel was detected by.Live/Dead experiment. The results were as follows: when the ultraviolet light time was 15,20,25,30 s, the strength of each hydrogel was about 13.87 + 1.30 Kpa, 19.76 + 1.61Kpa, 25.43 + 1.89Kpa, and 31.23 + 3.21Kpa.Presto Blue, which showed that the 10%PFTBA group was good. .Live/Dead fluorescence staining of the proliferation of hypoxia group cells showed that compared with the control group, there were more surviving cells in the 10%PFTBA group and the normal oxygen content group, and the cells spread well in the PFTBA-Gel MA hydrogel. Conclusion: the PFTBA-Gel MA hydrogel produced by this experimental method can adjust the mechanical strength of the hydrogel according to the ultraviolet light time. Degree; the hydrogel has good biocompatibility and can provide sufficient oxygen for cells under anoxic environment to maintain good cell activity. Experiment two: the influence of PFTBA-GelMA hydrogel on ADSCs osteogenesis and VECs vascularization under anoxic condition: after the fracture or bone defect occurred, the blood vessels in the damaged area were destroyed in a large amount. The method provides sufficient oxygen for the damaged area. There is a study that oxygen partial pressure is ~12.5% in normal bone. When fracture or bone defect occurs, oxygen content is rapidly reduced to ~1%, resulting in a large number of cells in the anoxic environment, causing cell necrosis and seriously affecting the repair of bone defects. And different oxygen pressure also seriously affects the function of the cells. It is reported that a large number of cell necrosis in the center of the scaffold is reported when the tissue engineering scaffold is in the low oxygen condition for a long time. It can be seen that oxygen supply after the fracture not only affects the regeneration of the fracture itself, but also affects the function of the tissue engineering scaffold. Therefore, the oxygen supply after bone defect is necessary. In this experiment, we use one. PFTBA-Gel MA hydrogel, a material that can release oxygen, was recombined with ADSCs and VECs to observe their effects on their osteogenesis and vascularization. Methods: PFTBA (0%, 5%, 10%) of different concentrations were integrated into Gel MA hydrogels, which were used as 3D culture scaffolds, and were combined into ADSCs, VECs respectively. Under the hypoxia environment (1%O2), the PFTBA pairs were observed. The effect of ADSCs osteogenesis and VECs vascularization; using ALP, alizarin red staining and immunofluorescence staining, to detect the early osteogenic differentiation of ADSCs and the formation of late calcium nodules. Observe the vascular situation of VECs and observe the length of the total vascular formation and the number of blood vessels, and study the vascularization of different groups of cells. To the ADSCs group and in the ADSCs group. The related genes were detected in VECs group (ALP, BMP-2, RUNX2, OPN; vascular related genes: CD31, VEGF). Co culture of ADSCs monolayer or VECs in proportion to 1:1 was carried out in a hypoxia environment, and the related genes were detected in the hypoxia environment, and the effect of PFTBA on monolayer cells and ADSCs-VECs co culture system was observed. Results: the results of ALP osteogenesis and alizarin red staining showed that the cells in the 10%PFTBA group had better osteogenesis, and OPN/F-actin fluorescence double staining also obtained similar results. Through the study of VECs angiogenesis, the ability of 10%PFTBA to form blood vessels after 12 h was found to be the best. Gene detection showed that in the 10%PFTBA group, the osteogenic related genes ALP, RUNX2, OPN were found in the 10%PFTBA group. The expression of BMP-2 was not different from that of the normal oxygen content group, which was significantly higher than that of the single layer hypoxia group and the 5%PFTBA group. Under the co culture system, the gene test showed that the co culture group could improve the osteogenic differentiation and vascularization better than the monolayer culture group. Conclusion: the 10%PFTBA-GelMA hydrogel can significantly promote ADSCs under the condition of oxygen deficiency. Osteogenic differentiation and VECs angiogenesis. This effect was further strengthened under co culture conditions. Experiment three: the internal research background of bone repair with composite ADSCs and VECs PFTBA-Gel MA hydrogel scaffolds: trauma, infection, tumor, osteomyelitis, debridement and other bone defects, autogenous bone graft, allograft bone graft, bioceramics, etc. It is possible for the repair of bone defects. However, it should be noted that the limited source of bone, immune rejection, biocompatibility and other factors restrict the application of these bone materials. The development of tissue engineering provides a new idea for the repair of bone defects. However, after the bone defect occurs, the damaged blood vessels in the defect area cannot be organized. The regenerative scaffold provides sufficient oxygen and restricts the self repair of the bone itself under the condition of hypoxia. Therefore, the solution of oxygen supply in the defect area has become an urgent problem. The combined ADSCs and VECs PFTBA-Gel MA hydrogels used in this study have been proved to be able to promote osteogenesis and vascularization in vitro. The purpose: To study the effect of the PFTBA-Gel MA hydrogel with ADSCs and VECs on the repair of ectopic osteogenesis and bone defect in the body. Methods: experimental groups: group Control, ADSCs+VECs group, Cells+PFTBA group. The hydrogels were transplanted into the nude mice on the back of the skin at.2 weeks, and the samples were stained for 4 weeks, and the ectopic was observed. Bone formation. All the hydrogels were transplanted into the bone defect of 5mm in the diameter of the rat's skull. The osteogenesis was detected by Micro-CT scanning at 2,4 weeks, and the gene detection of the experimental samples was carried out to detect the expression of the genes related to the osteogenesis and blood vessels. Results: the results of HE staining showed that the Cells+PFTBA group had a large number of bone matrix shapes. As compared with the Control group and the ADSCs+VECs group, the bone matrix formation was significantly increased by the.Micro-CT results. After 4 weeks, the new bone mass in the Cells+PFTBA group increased significantly compared with the Control and ADSCs+VECs groups. The gene detection showed that the expression of bone and vascularization related genes in Cells+PFTBA was significantly higher than that in the Control group. Conclusion: composite of Cells+PFTBA and vascularization related genes were significantly increased. PFTBA-Gel MA hydrogel scaffolds of ADSCs and VECs cells can promote ectopic osteogenesis and repair cranial bone defects in rats.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R68;R318.08
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本文編號(hào)：1814979