本文選題：殼聚糖　切入點(diǎn)：組織工程　出處：《山東大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：本課題旨在研制組織工程周圍神經(jīng),以解決長段神經(jīng)損傷修復(fù)中供體神經(jīng)來源缺乏問題。包括導(dǎo)管材料制備與表征、導(dǎo)管制作、神經(jīng)生長因子的固定化、種子細(xì)胞的分離培養(yǎng),并對(duì)所構(gòu)建的組織工程周圍神經(jīng)進(jìn)行了系統(tǒng)的功能評(píng)價(jià),以明確各影響因素在坐骨神經(jīng)損傷后修復(fù)過程中的作用。 殼聚糖(chitosan, CS)是一種天然的聚陽離子多糖,具有無毒、生物可降解、不會(huì)引起排異反應(yīng)等特點(diǎn)。但由于其脆性較高,不易加工,因而應(yīng)用受到一些限制。聚乳酸(polylactide, PLA)具有優(yōu)良的機(jī)械力學(xué)性能,對(duì)人體無毒、無刺激、可以被人體吸收,因而已被美國食品和藥品管理局(Food and Drug Administration,FDA)批準(zhǔn)用作藥物控釋載體及修復(fù)材料。但PLA存在親水性及組織相容性差,降解產(chǎn)物會(huì)引起局部酸性炎癥等不足。本課題采用接枝共聚法制備殼聚糖／聚乳酸的復(fù)合材料,以達(dá)到整合兩者優(yōu)點(diǎn),克服兩者不足的目的。 靜電自組裝(electrostatic self-assembly, ESA)技術(shù)已成為一種日益引起廣大生物材料工作者興趣的新型制備方法,它是基于相反電荷電解質(zhì)相互吸附的超薄膜形成技術(shù)。該技術(shù)操作簡單、成膜穩(wěn)定性好、不受基材形狀和大小的限制�，F(xiàn)己在生物材料改性、藥物緩釋、生物傳感器制備等領(lǐng)域廣泛應(yīng)用。本課題采用靜電自組裝技術(shù),將殼聚糖及肝素固定在5/0手術(shù)縫合線表面,制備神經(jīng)引導(dǎo)絲。并通過1-乙基-(3-二甲基氨基丙基)碳酰二亞胺鹽酸鹽(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC)交聯(lián)將神經(jīng)生長因子(nerve growth factor, NGF)固定于神經(jīng)引導(dǎo)絲及導(dǎo)管支架材料表面,以達(dá)到緩釋NGF的作用。 組織工程神經(jīng)導(dǎo)管的種子細(xì)胞中雪旺細(xì)胞是最理想的,其作用包括分泌多種神經(jīng)營養(yǎng)因子和細(xì)胞外基質(zhì),表達(dá)粘附分子,引導(dǎo)軸突再生等。但雪旺細(xì)胞本身是一種終末期細(xì)胞,體外培養(yǎng)的增殖能力差,很難達(dá)到移植所需的理想數(shù)量,且自體取材易造成新的損傷,故使其在臨床使用中受到一定的限制。骨髓來源的干細(xì)胞是較為理想的雪旺細(xì)胞替代細(xì)胞。本課題采用骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)作為種子細(xì)胞。 隨著交通方式的改變及機(jī)械化大生產(chǎn)的發(fā)展,當(dāng)前每年都有大量人員遭受周圍神經(jīng)損傷。顯微外科手術(shù)仍是現(xiàn)階段臨床上廣泛采用的治療手段,對(duì)于5mm以上的神經(jīng)損傷只能采用自體移植方法,但神經(jīng)來源有限,且會(huì)造成新的損傷。因此研究者開始將著眼于采用組織工程技術(shù)修復(fù)長距離的神經(jīng)損傷。該領(lǐng)域重點(diǎn)問題包括：①神經(jīng)導(dǎo)管材料選擇和表面形態(tài)修飾。②神經(jīng)導(dǎo)管與神經(jīng)營養(yǎng)因子的聯(lián)合應(yīng)用。③選擇并添加合適的種子細(xì)胞。本課題的工作即在此領(lǐng)域展開,主要包括： 1.殼聚糖／聚乳酸復(fù)合材料的構(gòu)建及性能研究 采用接枝共聚法制備殼聚糖／聚乳酸復(fù)合材料,并利用固態(tài)核磁、單反射紅外光譜、X射線衍射及掃描電子顯微鏡對(duì)其理化性質(zhì)進(jìn)行表征。測定殼聚糖氨基取代度、乳酸聚合度、孔隙率、溶脹性能、體內(nèi)外降解速率、體外降解pH變化及細(xì)胞親和性、組織相容性。結(jié)果表明,隨乳酸與殼聚糖質(zhì)量比的增大,復(fù)合材料孔隙率和溶脹性能逐漸減小,體外降解pH呈波動(dòng)性變化,且隨乳酸量的增大波動(dòng)性逐漸增大,體內(nèi)外降解質(zhì)量減少隨乳酸量的增大逐漸增大,在所設(shè)的三種比例下,復(fù)合材料的細(xì)胞親和性和組織相容性都較好。 2.組織工程神經(jīng)的構(gòu)建 2.1殼聚糖／聚乳酸復(fù)合材料神經(jīng)導(dǎo)管的制各 采用自制模具,利用冷凍干燥及高溫真空干燥方法制備復(fù)合材料導(dǎo)管支架。并對(duì)其力學(xué)性能及超微結(jié)構(gòu)進(jìn)行測定。結(jié)果表明,復(fù)合材料導(dǎo)管與殼聚糖導(dǎo)管相比可有效提高力學(xué)性能,且導(dǎo)管支架呈瓦片狀,能夠有效防止周圍組織的長入,同時(shí)有利于損傷神經(jīng)進(jìn)行物質(zhì)交換,能夠?yàn)樯窠?jīng)的再生提供良好的微環(huán)境。 2.2神經(jīng)引導(dǎo)絲的靜電自組裝修飾 采用可降解的聚乙醇酸5/0手術(shù)縫合線為載體,在其表面制備聚乳酸涂層,利用靜電自組裝技術(shù)在其表面制備殼聚糖／肝素多層膜結(jié)構(gòu),并通過紅外光譜、X射線光電子能譜及掃描電子顯微鏡進(jìn)行表征。結(jié)果表明,神經(jīng)引導(dǎo)絲表面已形成殼聚糖／肝素多層膜。并且在復(fù)合神經(jīng)導(dǎo)管后,可有效地增大導(dǎo)管的內(nèi)表面積。 2.3NGF的交聯(lián)固定 采用EDC交聯(lián)方式在神經(jīng)引導(dǎo)絲及導(dǎo)管支架上固定NGF,以達(dá)到在神經(jīng)修復(fù)期間緩釋NGF的作用。結(jié)果表明,經(jīng)釋放前期其呈現(xiàn)突釋,但隨時(shí)間的延長其逐漸呈平穩(wěn)釋放；且由于表面積大的原因,神經(jīng)導(dǎo)管固定NGF的量明顯多于神經(jīng)引導(dǎo)絲。 2.4BMSCs作為種子細(xì)胞的分離及熒光標(biāo)記 采用市售試劑盒,按照密度梯度離心法分離大鼠BMSCs與并與神經(jīng)導(dǎo)管復(fù)合構(gòu)建組織工程神經(jīng),應(yīng)用于大鼠坐骨神經(jīng)損傷修復(fù),采用Hoechst33258對(duì)BMSCs進(jìn)行標(biāo)記,結(jié)合免疫組化方法檢測其分化狀態(tài)。結(jié)果表明在手術(shù)6w后BMSCs依然存活,并且有nestin的表達(dá),表明其已經(jīng)分化為類神經(jīng)元細(xì)胞。 3.組織工程神經(jīng)對(duì)大鼠坐骨神經(jīng)損傷修復(fù)的效果觀察及初步機(jī)制研究 采用大鼠坐骨神經(jīng)損傷模型,以神經(jīng)電生理指數(shù),坐骨神經(jīng)功能指數(shù)(SFI),神經(jīng)的纖維密度、直徑及nestin免疫組化為考察指標(biāo),對(duì)比研究了空白組、造模組、自體移植組、組織工程神經(jīng)組、導(dǎo)管復(fù)合NGF組、導(dǎo)管復(fù)合干細(xì)胞組、固定有NGF的引導(dǎo)絲組、靜電自組裝修飾后的引導(dǎo)絲組、吡咯喹啉醌組、復(fù)合材料各組、殼聚糖組、聚乳酸組修復(fù)坐骨神經(jīng)損傷的效果,按效果從優(yōu)到劣的排序是：自體移植組、組織工程神經(jīng)組、NGF組、干細(xì)胞組、生長因子引導(dǎo)絲組、自組裝引導(dǎo)絲組、吡咯喹啉醌組、復(fù)合材料-Ⅱ組、復(fù)合材料-Ⅰ組、復(fù)合材料-Ⅲ組、殼聚糖組、聚乳酸組、造模組。熒光標(biāo)記和免疫組織化學(xué)檢測表明,nestin在組織工程神經(jīng)組及干細(xì)胞組均有表達(dá),表明BMSCs向神經(jīng)干細(xì)胞的方向進(jìn)行了分化,推測這是所構(gòu)建的組織工程神經(jīng)具有良好修復(fù)作用的機(jī)制。 本研究取得的主要研究成果有： 1.制備了殼聚糖／聚乳酸復(fù)合材料并對(duì)其理化性能,體內(nèi)外降解性能,細(xì)胞親和性及組織相容性進(jìn)行評(píng)價(jià),為制備適宜的組織工程材料提供依據(jù)。 2.利用靜電自組裝技術(shù)修飾神經(jīng)引導(dǎo)絲,在其表面制備肝素／殼聚糖膜,并對(duì)其理化性質(zhì)進(jìn)行表征；利用EDC交聯(lián)固定NGF,體外檢測顯示其可緩釋NGF；利用模具制作殼聚糖／聚乳酸復(fù)合材料支架。研究表明,復(fù)合材料-Ⅱ組的力學(xué)性能和微觀結(jié)構(gòu)更有利于損傷神經(jīng)的修復(fù)。 3.評(píng)價(jià)了各影響因素在大鼠周圍神經(jīng)損傷后修復(fù)中的效果。結(jié)果顯示各組修復(fù)情況排序?yàn)椋鹤泽w移植組、組織工程神經(jīng)組、NGF組、干細(xì)胞組、生長因子引導(dǎo)絲組、自組裝引導(dǎo)絲組、吡咯喹啉醌組、復(fù)合材料-Ⅱ組、復(fù)合材料-Ⅰ組、復(fù)合材料-Ⅲ組、殼聚糖組、聚乳酸組、造模組。結(jié)果表明,各影響因素的作用排序?yàn)椋篘GF、種子細(xì)胞、神經(jīng)引導(dǎo)絲、吡咯喹啉醌。 結(jié)果表明,通過接枝共聚法制備的殼聚糖/聚乳酸導(dǎo)管材料支架,在結(jié)合骨髓間充值干細(xì)胞及生長因子后,所制備組織工程周圍神經(jīng)對(duì)坐骨神經(jīng)損傷具有明顯的修復(fù)作用。
[Abstract]:This paper aims at the development of peripheral nerve tissue engineering, to solve the repair of long nerve injury in the donor nerve. The lack of sources including preparation and characterization, fabrication of catheter catheter materials, immobilized NGF, isolation and culture of seed cells, and to construct tissue-engineered peripheral nerves function evaluation system, in order to clear the influence factors in the process of repairing effect on sciatic nerve injury.
Chitosan (chitosan, CS) is a natural cationic polysaccharide, is non-toxic, biodegradable, will not cause the characteristics of rejection. But because of its high brittleness, easy processing, and the application of some restrictions. Polylactic acid (polylactide, PLA) has better mechanical properties, non-toxic to humans. No stimulation, can be absorbed by the body, which has been the United States Food and Drug Administration (Food and Drug Administration, FDA) for use as drug delivery carriers and repair materials. But PLA is hydrophilic and biocompatibility, degradation products can cause local inflammation. The acid was prepared by graft copolymerization the composite chitosan / poly lactic acid, in order to achieve the integration of the two advantages, to overcome the deficiencies of the two.
Electrostatic self-assembly (electrostatic self-assembly ESA) technology has become a new business has attracted considerable interest in biological material workers preparation method, it is the formation of thin film technology based on the mutual adsorption of oppositely charged electrolyte. This technology has the advantages of simple operation, good film-forming stability, is not affected by the shape and size of the substrate is in the limit. Instead, biological materials, drug delivery, wide application of biosensor and other fields. This paper adopts electrostatic self-assembly technology, chitosan and heparin immobilized on 5/0 suture surface, preparation of nerve guidance wire. And through 1- ethyl - (3- methyl propyl two amino carbonyl imine hydrochloride (two) (1- 3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC) the crosslinking of nerve growth factor (nerve growth, factor, NGF) is fixed on the guide wire and catheter surface nerve scaffold, in order to achieve the sustained release effect of NGF.
Schwann cell seed cells in tissue engineering nerve conduit is the most ideal, including its role in secretion of various neurotrophic factors and extracellular matrix, the expression of adhesion molecules, and axon regeneration. But Schwann cell itself is a kind of end-stage cells, the proliferation ability is poor in vitro, it is difficult to achieve the desired number required for transplantation, and autografts are apt to cause new damage, so it is limited in clinical use. Bone marrow stem cells is an ideal alternative to Schwann cells cells. The bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) as seed cells.
With the development and change of mechanized production mode of transportation, the current year a large number of peripheral nerve injury. Microsurgery is widely used at this stage is still in clinical treatment, only for more than 5mm of nerve injury by autologous transplantation method, but the nerve is limited and will cause new damage. Therefore researchers began to focus on using tissue engineering techniques for repairing nerve injury in long distance. The main issues in the field include: nerve conduit materials selection and modification of surface morphology. In combination of nerve conduit with neurotrophic factor. The select and add suitable seed cells. This research work in this field, including:
Study on the construction and properties of 1. chitosan / polylactic acid composites
Preparation of chitosan / polylactic acid composite material prepared by graft copolymerization, and the use of solid-state NMR, single reflection infrared spectroscopy, X ray diffraction and scanning electron microscope on its physicochemical properties were characterized. The determination of chitosan amino substitution degree, degree of polymerization of lactic acid, porosity, swelling properties, in vitro degradation rate, degradation in vitro and pH changes cell affinity, tissue compatibility. The results show that with the increase of the quality of lactic acid and chitosan ratio, composite porosity and swelling properties decreased gradually, in vitro degradation of pH fluctuated, with the increase in volatility increases the amount of lactic acid, reduced with the increase of the quality of in vivo and in vitro degradation of lactic acid increased gradually, in three the proportion of the composite material, cell affinity and tissue compatibility are good.
2. construction of tissue engineering nerve
The production of 2.1 chitosan / polylactic acid composite nerve conduits
Using homemade mould for drying and high temperature vacuum drying method using frozen composite material conduit. And the mechanical properties and ultrastructure were measured. The results show that the composite chitosan catheter and catheter compared can effectively improve the mechanical properties, and the conduit a shingle, can effectively prevent the surrounding tissue ingrowth, at the same time to damage nerve material exchange, can provide a good microenvironment for nerve regeneration.
Self assembling decoration of 2.2 nerve guide wire
The use of biodegradable polyglycolic acid 5/0 suture as the carrier, on the surface of the preparation of polylactic acid coating, electrostatic self-assembly technique to prepare chitosan / heparin multilayer film structure on the surface, and through the infrared spectrum, X ray photoelectron spectroscopy and scanning electron microscopy were characterized. The results showed that the nerve guide wire the surface has formed chitosan / heparin multilayer film. And the composite nerve conduit, can effectively increase the tube surface area.
Crosslinking fixation of 2.3NGF
The EDC guide wire and catheter cross bracket is fixed in NGF to achieve the function of nerve, release NGF in nerve repair period. The results showed that the burst release was released early, but with time prolonging the gradually stable release; and because the table causes a large area, nerve conduit was significantly more than the fixed NGF nerve guide wire.
Isolation and fluorescence labeling of 2.4BMSCs as seed cells
Using commercially available kits, in accordance with the density gradient centrifugation of rat with BMSCs and composite nerve conduit in nerve tissue engineering, applied to the repair of sciatic nerve injury in rats, using Hoechst33258 to mark BMSCs, combined with the immunohistochemical method to detect the differentiation status. Results show that the BMSCs is still alive in 6W after surgery, and the expression of nestin, indicating that it has differentiated into neuron like cells.
The effect of 3. tissue engineering nerve on the repair of sciatic nerve injury in rats and its preliminary mechanism
The rat model of sciatic nerve injury, the nerve electrophysiological index, sciatic nerve function index (SFI), nerve fiber density, diameter and nestin immunohistochemistry as investigation index, comparative study of the blank group, model group, autograft group and nerve tissue engineering composite duct group, NGF group, catheter composite stem the cell group, fixed with NGF guide wire group, guide wire group electrostatic self-assembly of pyrroloquinoline quinone group, composite group, chitosan group, polylactic acid group and repair of sciatic nerve injury effect, according to the effect in descending order: autograft group, tissue engineering nerve group, NGF group, stem cell growth factor group, guide wire group, self assembling guide wire group, pyrroloquinoline quinone group, composite II group, composite material and composite material - I group, III group, chitosan group, poly lactic acid group, model group showed fluorescence labeling and immunohistochemical detection of nestin. It is expressed in tissue-engineered nerve and stem cell groups, indicating that BMSCs has differentiated into the direction of neural stem cells. It is presumed that this tissue engineering nerve has good repair effect.
The main achievements of this study are as follows:
1., chitosan / PLA composites were prepared, and their physicochemical properties, in vitro and in vivo degradability, cell affinity and histocompatibility were evaluated, providing a basis for preparing suitable tissue engineering materials.
2. using the electrostatic self-assembly technique modified microfilament, preparation of heparin / chitosan film on its surface, and physicochemical properties were characterized by EDC; cross-linked NGF, in vitro testing showed that the sustained-release NGF; making chitosan / polylactic acid composite scaffold using a mold. The research shows that the mechanical properties and the microstructure is more composite materials of group II to the repair of nerve injury.
3. evaluation of various factors in the repair of peripheral nerve injury in rats. The results showed the repair order: autograft group, tissue engineering nerve group, NGF group, stem cell growth factor group, guide wire group, self assembling guide wire group, pyrroloquinoline quinone group, composite II group composite materials, composite materials - - I group, III group, chitosan group, poly lactic acid group and model group. The results show that the influence of the order: NGF, seed cells, nerve guide wire, pyrroloquinoline quinone.
The results showed that the chitosan / polylactic acid catheter scaffolds prepared by graft copolymerization combined with bone marrow restores stem cells and growth factors, and the tissue-engineered peripheral nerve tissue had significant repair effect on sciatic nerve injury.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08

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