本文選題：生物材料 + 干細(xì)胞��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：生物材料是一類(lèi)目的在于和生物系統(tǒng)發(fā)生相互作用,來(lái)評(píng)價(jià)、治療、增強(qiáng)或替代某種組織、器官或者人體功能的材料。理解和認(rèn)識(shí)生物材料-細(xì)胞界面以及優(yōu)化和設(shè)計(jì)生物材料的結(jié)構(gòu)和性質(zhì),將對(duì)構(gòu)建細(xì)胞外微環(huán)境以及生物醫(yī)學(xué)工程的具體實(shí)施具有重要的指導(dǎo)意義。本文針對(duì)目前廣泛關(guān)注的不同類(lèi)型的微環(huán)境中材料學(xué)信號(hào)對(duì)細(xì)胞的作用進(jìn)行了研究,其中包括,材料表面電荷微環(huán)境、納米顆粒微環(huán)境、生物因子微環(huán)境與仿生細(xì)胞外基質(zhì)微環(huán)境,目的是揭示材料表面性質(zhì)和細(xì)胞外微環(huán)境和細(xì)胞行為的相互作用,為生物材料設(shè)計(jì)和制備奠定理論和實(shí)驗(yàn)基礎(chǔ)。本文的研究主要從以下幾個(gè)方面展開(kāi)：1.材料表面電荷對(duì)干細(xì)胞行為的影響：生物材料的表面物理性質(zhì)和生物化學(xué)性質(zhì)對(duì)細(xì)胞的貼壁、遷移、增殖和分化等行為有很大的影響。但是在調(diào)查單個(gè)物理或化學(xué)因素與細(xì)胞的相互作用時(shí),很多研究并不能在單一變量的體系下進(jìn)行。在單因素變量的二維模型中研究材料表面性質(zhì)對(duì)細(xì)胞行為的影響可以更清楚的認(rèn)識(shí)和理解細(xì)胞對(duì)材料表面性質(zhì)的響應(yīng)。生物材料表面電荷是影響細(xì)胞行為的重要因素,但目前關(guān)于干細(xì)胞對(duì)表面電荷的響應(yīng)尤其是表面電荷對(duì)干細(xì)胞分化的影響的研究很少。因此,本論文首次利用不同切割方向的鈮酸鋰單晶薄片構(gòu)建化學(xué)成分相同、表面粗糙度相同的但具有不同電荷表面的單變量簡(jiǎn)單模型,并用其作為二維培養(yǎng)基底來(lái)研究材料表面不同電荷狀態(tài)對(duì)間充質(zhì)干細(xì)胞的貼壁、增殖以及成骨分化的影響。論文證實(shí)了相比于負(fù)電和中性表面,帶正電荷的表面可以促進(jìn)細(xì)胞貼壁鋪展以及在體外誘導(dǎo)條件下增強(qiáng)大鼠間充質(zhì)干細(xì)胞向成骨分化。2.細(xì)胞外微環(huán)境中納米晶體對(duì)干細(xì)胞的影響：干細(xì)胞在生物醫(yī)學(xué)尤其是再生領(lǐng)域的應(yīng)用仍然面臨著重大挑戰(zhàn),這包括發(fā)展先進(jìn)技術(shù)來(lái)理解和控制微環(huán)境中的各種信號(hào),以及新的手段來(lái)追蹤和引導(dǎo)植入的干細(xì)胞。納米材料在分子水平上和細(xì)胞特異性的作用是理解和控制干細(xì)胞的行為的重要工具。比如基于納米顆粒的載藥技術(shù)可以靶向釋放細(xì)胞所需的活性因子,靜電紡絲技術(shù)合成的納米纖維支架可以引導(dǎo)細(xì)胞的生長(zhǎng)及組織形成,以及使用量子點(diǎn)和上轉(zhuǎn)化等納米材料進(jìn)行細(xì)胞成像和追蹤。隨著這些大量功能性的納米材料在生物醫(yī)學(xué)工程上的應(yīng)用,干細(xì)胞對(duì)這些人為引入到微環(huán)境中的納米顆粒的響應(yīng)需要確認(rèn)和研究。本論文利用溶劑熱的方法制備了鈮酸鋰納米晶體,利用NIR飛秒激光器作為激發(fā)光源在雙光子顯微鏡下觀察大鼠間充質(zhì)干細(xì)胞對(duì)微環(huán)境中鈮酸鋰納米晶體的主動(dòng)內(nèi)化吞噬,以及利用PCR和免疫染色等生物學(xué)手段表征鈮酸鋰納米晶體對(duì)干細(xì)胞分化的影響。論文證實(shí)了干細(xì)胞對(duì)微環(huán)境中納米晶體的主動(dòng)響應(yīng)和分化調(diào)控。3.通過(guò)生長(zhǎng)因子的緩釋構(gòu)建組織修復(fù)生物微環(huán)境：控制細(xì)胞行為的細(xì)胞外組成不僅包括如上所述的細(xì)胞外基質(zhì)中不可溶的成分,還包括了激素和生長(zhǎng)因子等可溶性的分子。所以除了利用生物材料本身表面和結(jié)構(gòu)性質(zhì)指導(dǎo)細(xì)胞行為,生物材料結(jié)合可溶性的生長(zhǎng)因子來(lái)控制細(xì)胞分化和功能也是一個(gè)構(gòu)建細(xì)胞外微環(huán)境的重要手段。目前,外科手術(shù)修復(fù)肌腱等結(jié)締組織仍舊是臨床上一個(gè)挑戰(zhàn),缺損處組織修復(fù)后很難再達(dá)到原來(lái)的強(qiáng)度水平。為了在修復(fù)前期給缺損處提供初始的力學(xué)支持,人們嘗試通過(guò)增強(qiáng)縫合線的強(qiáng)度以及改進(jìn)縫合線的抓握方法。雖然這些方法能夠一定程度上幫助組織修復(fù),但是這些方法并不能為組織修復(fù)創(chuàng)造合適的微環(huán)境,也不能調(diào)節(jié)修復(fù)的生物過(guò)程。因此,本論文首次提出基于多孔縫合線的生長(zhǎng)因子緩釋體系來(lái)構(gòu)建組織修復(fù)的微環(huán)境。研究使用一種簡(jiǎn)單有效的溶脹-冷凍-干燥方法來(lái)制備具有多孔結(jié)構(gòu)鞘層的縫合線而不降低其原有的機(jī)械強(qiáng)度。由于多孔結(jié)構(gòu)的存在,縫合線內(nèi)部的空間就可以被利用起來(lái)以實(shí)現(xiàn)更高的負(fù)載。另外,多孔結(jié)構(gòu)結(jié)合使用纖維蛋白交聯(lián)網(wǎng)絡(luò)能減緩后續(xù)的釋放過(guò)程,研究實(shí)現(xiàn)了PDGF在體外生理環(huán)境中長(zhǎng)達(dá)11天的持續(xù)釋放。而且釋放的PDGF保持了原有的生物活性,能夠促進(jìn)人類(lèi)骨髓間充質(zhì)干細(xì)胞的增殖。這種基于多孔縫合線的生長(zhǎng)因子緩釋體系為構(gòu)建組織修復(fù)微環(huán)境提供了新的策略。4.利用天然細(xì)胞外基質(zhì)仿生構(gòu)建細(xì)胞外微環(huán)境的探索：細(xì)胞外基質(zhì)(ECM)是一個(gè)結(jié)構(gòu)和功能都很復(fù)雜的混合物,在細(xì)胞的行為和表型上起著非常重要的作用,因此人們努力嘗試希望能得到一個(gè)人造的ECM等價(jià)物,來(lái)給細(xì)胞提供一個(gè)模擬天然的微環(huán)境。將組織的細(xì)胞成分脫除得到的脫細(xì)胞基質(zhì)已經(jīng)越來(lái)越多的被用于組織再生和器官移植等再生醫(yī)學(xué)領(lǐng)域。但是由于天然的細(xì)胞外基質(zhì)主要成分是膠原,支架材料的機(jī)械強(qiáng)度較低并且降解速率較快,這制約了其在組織工程尤其是骨組織工程上的應(yīng)用。因此,本論文將使用優(yōu)化的脫細(xì)胞技術(shù)制備豬脫細(xì)胞真皮基質(zhì)(PADM),并使用碳化二亞胺作為交聯(lián)劑強(qiáng)化PADM,以此嘗試模擬細(xì)胞外基質(zhì)微環(huán)境。得到的PADM支架保留了膠原天然的多孔結(jié)構(gòu),并有效保留了膠原和GAG等有效成分,去除了核酸和SDS等免疫反應(yīng)源；評(píng)價(jià)了碳化二亞胺作為零長(zhǎng)度交聯(lián)劑對(duì)PADM支架的交聯(lián)作用,及交聯(lián)對(duì)支架強(qiáng)度和降解速率的影響；體外評(píng)估了前成骨細(xì)胞在交聯(lián)后PADM支架的生物活性。交聯(lián)后的PADM的力學(xué)強(qiáng)度和降解速率都可以通過(guò)交聯(lián)程度控制,作為仿生支架模擬細(xì)胞外微環(huán)境也可以很好地支持細(xì)胞向支架內(nèi)部遷移。因此,本研究為使用天然細(xì)胞外基質(zhì)來(lái)模擬細(xì)胞外基質(zhì)微環(huán)境的探索提出了一種可能性。綜上所述,本論文主要致力于研究生物材料表面對(duì)細(xì)胞行為的調(diào)控以及探索細(xì)胞外微環(huán)境的體外構(gòu)建,這些研究和探索將進(jìn)一步促進(jìn)生物材料在生物醫(yī)學(xué)工程上的應(yīng)用。
[Abstract]:Biomaterials are a class of materials that aim to interact with biological systems to evaluate, treat, enhance or replace some tissues, organs, or human functions. Understanding and understanding the biomaterial cell interface and optimizing and designing the structure and properties of biological materials will be for the construction of the extracellular microenvironment and biomedical engineering. In this paper, the effect of material signal on the cell is studied in different types of microenvironment, including the surface charge micro environment, nano particle microenvironment, biological factor microenvironment and biomimetic extracellular matrix microenvironment. The purpose is to reveal the surface properties of the material. The interaction between the extracellular microenvironment and cell behavior will lay a theoretical and experimental basis for the design and preparation of biomaterials. This study mainly starts from the following aspects: 1. the effect of surface charge on the behavior of stem cells: the surface physical properties and biochemical properties of biomaterials on cell adhesion, migration, and proliferation. But in investigating the interaction of single physical or chemical factors with cells, many studies can not be carried out in a single variable system. In the two-dimensional model of single factor variables, the study of the effect of material surface properties on cell behavior can be more clearly understood and understood. Response of surface properties. Surface charge of biomaterials is an important factor affecting cell behavior, but there are few studies on the effect of stem cells on surface charge, especially surface charge, on the differentiation of stem cells. Therefore, the first use of different cutting direction lithium niobate single crystal slices to construct the same chemical composition and surface roughness in this paper. A simple single variable model with the same degree but with a different charge surface, and using it as a two-dimensional culture substrate to study the effect of different charge states on the adhesion, proliferation and osteogenesis of mesenchymal stem cells on the surface of the material. And the effect of nanocrystalline on stem cells in the extracellular microenvironment of osteogenic differentiation.2. in vitro induced by rat mesenchymal stem cells: the application of stem cells in biomedicine, especially in the field of regeneration, still faces major challenges, including the development of advanced technology to understand and control the various signals in microenvironment, and the new technology. The means to track and guide implanted stem cells. The role of nanomaterials at molecular level and cell specificity is an important tool for understanding and controlling the behavior of stem cells. For example, nanoparticles based drug loading techniques can target the active factors needed to release the cells, and the nanofiber scaffolds synthesized by the electrospun spinning technology can be guided. Cell growth and tissue formation, and the use of nanomaterials, such as quantum dots and upper conversion, to carry out cell imaging and tracking. With the application of these large functional nanomaterials in biomedical engineering, the response of the stem cells to nano particles introduced into the microenvironment should be confirmed and studied. This paper uses solvent heat. Lithium niobate nanocrystals were prepared by using a NIR femtosecond laser as an excitation source to observe the active endogenous phagocytosis of the rat mesenchymal stem cells to the lithium niobate nanocrystals in microenvironment under two photon microscopy, and the effects of PCR and immunologic staining on the differentiation of the stem cells by using PCR and immunologic staining. The active response and differentiation of nanocrystals in the microenvironment are regulated by stem cells to repair the microenvironment of.3. through the sustained release of growth factors. The extracellular components that control cell behavior include not only the insoluble components in the extracellular matrix as described above, but also the soluble fractions such as hormones and growth factors. So in addition to using the surface and structural properties of biological materials to guide cell behavior, biological materials combine soluble growth factors to control cell differentiation and function is an important means to construct extracellular microenvironment. At present, surgical repair of connective tissue such as tendons remains a clinical challenge, and the defect is repaired in tissue. It is difficult to reach the original strength level. In order to provide initial mechanical support to the defect in the early period of repair, people try to enhance the strength of the suture line and improve the grip of the suture. Although these methods can help the tissue repair to a certain extent, these methods do not create suitable for tissue repair. Microenvironment can not regulate the biological process of repair. Therefore, this paper first proposed a sustained release system of growth factor based on porous suture to construct microenvironment for tissue repair. A simple and effective method of swelling freeze drying was used to prepare suture with porous structure sheath without reducing its original mechanical strength. Due to the existence of the porous structure, the space inside the suture can be used to achieve higher load. In addition, the porous structure combined with fibrin crosslinking network can slow down the subsequent release process. The study realized the sustained release of PDGF for up to 11 days in the physiological environment in vitro. And the release of PDGF maintained the original life. The activity of substance can promote the proliferation of human bone marrow mesenchymal stem cells. This growth factor sustained-release system based on porous suture provides a new strategy for the construction of microenvironment for tissue repair. The extracellular matrix (ECM) of the extracellular matrix (.4.) is a complex structure and function. The mixture plays a very important role in the behavior and phenotype of cells, so people try to try to get a person's ECM equivalent to provide a simulated natural microenvironment. The more the cell components removed from the tissue, the more the cells are used for tissue regeneration and organ transplantation. But because the natural extracellular matrix is mainly collagen, the mechanical strength of the scaffold material is low and the degradation rate is fast, which restricts its application in tissue engineering especially bone tissue engineering. Therefore, this paper will use the optimized acellular technique to prepare the porcine acellular dermal matrix (PADM), and use it in this paper. As a crosslinker, carbonized two imide was used as a crosslinker to simulate the microenvironment of extracellular matrix. The PADM scaffold retained the natural porous structure of collagen, retained effective components such as collagen and GAG, removed the immune response sources such as nucleic acid and SDS, and evaluated the crosslinking of PADM as a zero length cross-linking agent as a zero length cross-linking agent. The effect of interaction and cross-linking on the strength and degradation rate of the scaffold; in vitro evaluation of the bioactivity of the PADM scaffold after cross linking of osteoblasts. The mechanical strength and degradation rate of the crosslinked PADM can be controlled by the degree of cross-linking. As a biomimetic scaffold, the extracellular microenvironment can also be well supported by the migration of the cell to the stent. Therefore, this study provides a possibility for the use of natural extracellular matrix to simulate the microenvironment of extracellular matrix. In summary, this paper focuses on the study of the regulation of cell behavior on the surface of biomaterials and the exploration of the external microenvironment in vitro. These research and exploration will further promote biological materials. Biomedical engineering applications.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R318.08

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