ADSCs-VECs复合携氧材料PFTBA-GelMA水凝胶促进骨缺损修复的研究

发布时间：2018-04-28 10:59

  本文选题：PFTBA + GelMA水凝胶　； 参考：《第四军医大学》2017年博士论文

【摘要】：严重骨折和骨缺损导致骨的连续性和结构完整性被破坏,导致患肢功能受损甚至丧失功能,严重影响着患者的生活质量,是骨科领域急需解决的难题之一。骨内血管分布较为丰富,为骨组织正常的功能运转提供相应的氧气和营养成分。大量研究表明:骨折后,骨折处血管遭到大量破坏,使受损处组织的氧份和营养供应不足,严重影响骨折后的修复。如果缺损过大(≥8 mm),其自身很难完全愈合,易造成骨延迟愈合或骨不连等并发症的发生。组织工程和生物材料技术的应用为骨修复提供了新的方法。但是,应当注意到在缺氧条件下,种子细胞的存活和相应的功能发挥受到极大的抑制,这影响了其修复骨缺损的效能。因此,严重骨折和骨缺损后,缺损区域的氧气供应对骨修复十分重要。现如今有很多携氧和释放氧气的材料被用于缺氧的研究。全氟三丁胺(Perfluorotributylamine,PFTBA)因其具有较好的氧溶解性和无毒副作用,被广泛用于解决组织缺氧的研究中。但是,PFTBA释放氧气的时间为4-5天,因此如果将PFTBA应用于骨缺损的修复,还需考虑4-5天后的氧气供应。新生血管可以为骨缺损处提供氧气,血管组织分为三层,分别为内膜、中膜和外膜,其中最里层的内皮细胞对新生血管的形成起着关键的作用。如果将此细胞应用于骨缺损的修复,可以加速缺损处的血管化进程。因此为更好地促进骨折或骨缺损后的骨修复,本研究尝试制做一种复合有携氧材料PFTBA的甲基丙烯酸酯明胶Gelatin methacryloyl(Gel MA)支架材料,并将脂肪干细胞与血管内皮细胞复合进此材料,用于严重骨折或者骨缺损修复的相关研究,以期在骨缺损的修复过程中为其提供充足的氧气且加速骨折处的血管再生,促进受损区骨组织更快更好的修复。整个研究分为以下三个部分:实验一:PFTBA-Gel MA水凝胶支架的制备及相关理化性质和生物性能检测背景:严重骨折和骨缺损导致患肢功能受限甚至丧失功能,严重影响着患者的生活质量。自体或异体骨移植是治疗骨缺损的最有效的办法,但是由于骨的供源不足,取骨过程易造成二次损伤,以及异体骨产生的免疫排斥反应,均限制了其应用。组织工程技术的发展为解决这一难题提供了新的方法。载体生物支架作为组织工程的三要素之一,应具有良好的生物相容性和可降解性,能够促进细胞的存活,增殖,分化,黏附,另外有研究指出不同机械强度的细胞微环境能促进细胞产生不同的功能应答,因此该生物支架应该具有可调节的机械力学性能。同时,在体内,细胞均立体生活在相应的生物基质内。模拟细胞的生存环境对细胞功能的发挥十分有利,现如今大量水凝胶材料应用于细胞的三维培养,包括胶原蛋白(Collagen),藻酸盐(Alginate),透明质酸(Hyaluronic acid,HA),Gel MA等。其中,Gel MA水凝胶材料以其易合成,造价低,生物相容性较好,可调节机械强度等优点被广泛应用于组织工程领域。骨缺损造成的低氧环境不利于骨组织的修复。PFTBA是一种很好的携氧材料,在本实验中,我们将其与Gel MA混合,制作出PFTBA-Gel MA水凝胶,以期在应用中释放足够的氧气为骨修复所用。目的:构建生物相容性较好的PFTBA-Gel MA水凝胶材料,并检测其理化性质和生物学性能。方法:采用化学合成的方法制作出Gel MA水凝胶,将不同浓度的PFTBA(0,5%,10%)整合进Gel MA水凝胶内,采用不同的紫外光照时间交联,制作出不同硬度的水凝胶材料。用Instron机器来检测各组水凝胶的杨氏模量。以此水凝胶为3D培养支架,复合进ADSCs,缺氧环境下(1%O2)观察该PFTBA-Gel MA水凝胶对ADSCs细胞活性的影响(Presto Blue实验)。Live/Dead实验检测细胞在该水凝胶内的存活状态,统计其存活细胞率。结果:当紫外光照时间分别为15,20,25,30 s时,各组水凝胶的强度分别约为13.87±1.30 Kpa,19.76±1.61Kpa,25.43±1.89Kpa,31.23±3.21Kpa。Presto Blue结果表明,10%PFTBA组能够很好的促进缺氧组细胞的增殖。Live/Dead荧光染色显示:相比对照组,10%PFTBA组和正常氧含量组存活的细胞较多,且细胞在PFTBA-Gel MA水凝胶里伸展较好。结论:通过本实验方法制作的PFTBA-Gel MA水凝胶能够根据紫外光照时间来调节水凝胶的机械强度;该水凝胶具有良好的生物相容性,能为缺氧环境下的细胞提供足够的氧气,使其保持较好的细胞活性。实验二:缺氧条件下,PFTBA-GelMA水凝胶对ADSCs成骨分化和VECs血管化的影响背景:骨折或骨缺损发生后,受损区域血管遭到大量破坏,无法为受损区域输送足够的氧气,有研究指出氧分压在正常骨的含量为~12.5%,当骨折或骨缺损发生时,氧含量迅速降到~1%,造成大量细胞的处于缺氧环境,引起细胞坏死,严重影响了骨缺损后的修复。且不同氧分压也严重影响着细胞的功能发挥。据报道,当组织工程支架长期处于低氧条件下时,会造成支架中心大量的细胞坏死。可见,骨折后的氧供应不仅影响着骨折本身的再生,还影响着组织工程支架功能的发挥。因此,骨缺损后的氧供应十分必要。在本实验中我们使用一种能够释放氧气的材料PFTBA-Gel MA水凝胶,再复合以ADSCs和VECs,观察其对它们成骨和血管化的影响。方法:将不同浓度的PFTBA(0%,5%,10%)整合进Gel MA水凝胶内,以此体系为3D培养支架,分别复合进ADSCs,VECs,并将其放置缺氧环境(1%O2)下,观察PFTBA对ADSCs成骨分化,VECs血管化的影响;通过ALP,茜素红染色和免疫荧光染色,检测ADSCs的早期成骨分化,和晚期钙结节的形成情况。对VECs成血管情况进行观察,并对总血管形成长度和血管数量进行统计,研究不同组细胞的血管化情况。对ADSCs组和VECs组分别进行相关基因检测(成骨相关基因:ALP,BMP-2,RUNX2,OPN;成血管相关基因:CD31,VEGF)。将ADSCs单层或者和VECs按1:1比例进行共培养,分别将其置于缺氧环境下,对其进行相关基因检测,观察PFTBA对单层细胞,和ADSCs-VECs共培养体系的作用。结果:ALP成骨染色和茜素红染色结果显示10%PFTBA组的细胞成骨分化效果较好,OPN/F-actin荧光双标染色也得到了类似的结果。通过对VECs成血管进行研究,发现12 h后10%PFTBA组成血管的能力最好。基因检测表明在10%PFTBA组,成骨相关基因ALP,RUNX2,OPN,BMP-2的表达和正常氧含量组无差异,和单层缺氧组和5%PFTBA组相比显著提高。在共培养体系下,基因检测表明10%PFTBA条件下,相比单层培养组,共培养组能更好的促进成骨分化和血管化。结论:缺氧条件下,10%PFTBA-GelMA水凝胶能显著促进ADSCs的成骨分化和VECs的成血管。此作用在共培养条件下得到进一步加强。实验三:复合ADSCs和VECs的PFTBA-Gel MA水凝胶支架对骨修复的体内研究背景:创伤、感染、肿瘤、骨髓炎手术清创等易导致骨缺损的发生,自体骨移植,异体骨移植,生物陶瓷等应用为骨缺损的修复提供了可能。但是,同时应注意到骨来源有限,免疫排斥反应,生物相容性等因素限制了这些骨材料的应用。组织工程的发展为骨缺损的修复提供了新的思路。但是,骨缺损发生后,缺损区域遭到破坏的血管无法为组织再生支架提供足够的氧气,且缺氧条件下还限制骨本身的自我修复。因此,解决缺损区域的氧供应成为了目前一项亟需解决的问题。本研究采用的复合ADSCs和VECs的PFTBA-Gel MA水凝胶已被证明能够很好地在体外促进成骨分化和血管化,但是在体内是否有效还未得到证实。目的:研究复合ADSCs和VECs的PFTBA-Gel MA水凝胶对体内异位成骨和骨缺损的修复作用。方法:实验分组:Control组,ADSCs+VECs组,Cells+PFTBA组。将各组水凝胶分别移植入裸鼠背部皮下。2周,4周取材进行HE染色,观察异位成骨情况。将各组水凝胶移植入大鼠颅骨直径为5mm的骨缺损。分别在2,4周取材,进行Micro-CT扫描检测,检测其成骨能力。对实验样本进行基因检测,检测其成骨和成血管相关基因的表达情况。结果:HE染色结果显示Cells+PFTBA组有大量骨基质形成,相比Control组和ADSCs+VECs组,骨基质的形成量显著增加。Micro-CT结果表明,4周后,与Control组和ADSCs+VECs组相比,Cells+PFTBA组的新生骨量显著增多。基因检测表明,与Control组相比,Cells+PFTBA组成骨和血管化相关基因的表达均显著上升。结论:复合ADSCs和VECs细胞的PFTBA-Gel MA水凝胶支架能够促进异位成骨,并能较快的修复大鼠颅骨骨缺损。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68;R318.08
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本文编号：1814979