本文選題：軟骨微粒 + 仿生基質(zhì)溶膠��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：背景:關(guān)節(jié)軟骨損傷臨床發(fā)病率越來越高,正被人們高度關(guān)注。由于正常透明軟骨是一種特殊組織,無血管、神經(jīng),損傷后其自我修復(fù)能力低下,直徑超過4mm的軟骨缺損不能完全自我修復(fù)。軟骨損傷后,會(huì)導(dǎo)致關(guān)節(jié)腫脹和疼痛,影響運(yùn)動(dòng)功能,不及時(shí)治療,最終發(fā)展為骨性關(guān)節(jié)炎。傳統(tǒng)治療關(guān)節(jié)軟骨缺損的方法有:微骨折技術(shù)、自體/異體骨軟骨移植技術(shù)等,但都存在一定的缺陷和不足。隨著組織工程技術(shù)的發(fā)展,自體軟骨細(xì)胞移植(ACI)技術(shù)在臨床得到應(yīng)用,并取得明顯治療效果,但還是存在明顯缺陷:首先軟骨細(xì)胞體外培養(yǎng)、擴(kuò)增,難以模擬在體微環(huán)境,缺乏機(jī)體免疫系統(tǒng)的監(jiān)控,植入的軟骨細(xì)胞通常已經(jīng)發(fā)生表型改變,最終形成的修復(fù)組織無論在結(jié)構(gòu)上還是成份上都與天然軟骨存在本質(zhì)差異,其次該方法需要二次手術(shù)植入修復(fù)缺損,治療期限跨度長(zhǎng),身體損傷大,給患者帶來巨大身體痛苦和經(jīng)濟(jì)負(fù)擔(dān)。目的:本研究利用組織工程技術(shù)體外構(gòu)建組織工程軟骨,無菌條件下取貴州小香豬膝關(guān)節(jié)軟骨,制作成軟骨微粒,體外培養(yǎng)后觀察軟骨微粒中軟骨細(xì)胞脫落、增殖情況,篩選最佳增殖效果的粒徑范圍,然后用增殖效果最佳的軟骨微粒作為種子細(xì)胞;制備Ⅱ型膠原蛋白、硫酸軟骨素、透明質(zhì)酸(COLⅡ/CS/HA)仿生基質(zhì)凝膠,加入EDAC交聯(lián)劑,升溫成凝膠后,檢測(cè)其成膠性能;用軟骨微粒與仿生基質(zhì)溶膠混合后體外共培養(yǎng),通過大體和組織學(xué)檢測(cè),評(píng)估體外構(gòu)建組織工程軟骨的可行性。本實(shí)驗(yàn)成功后,可望為一次手術(shù)治療關(guān)節(jié)軟骨缺損提供依據(jù)。方法:取3-5月齡貴州小香豬膝關(guān)節(jié),把軟骨制成微粒狀;將微粒用孔徑分別為120μm、212μm、475μm、880μm微粒篩過濾;收集不同粒徑的軟骨微粒,按粒徑由小至大設(shè)A、B、C、D組,保證每組質(zhì)量相同,均勻種植到3個(gè)12孔培養(yǎng)板中,用高糖培養(yǎng)基培養(yǎng);培養(yǎng)1d、3d、7d時(shí),分別取出1板進(jìn)行細(xì)胞計(jì)數(shù),篩選脫落、增殖效果最佳的粒徑范圍。用環(huán)鉆在離體新鮮豬膝關(guān)節(jié)上制備直徑8mm全厚軟骨缺損模型;將COLⅡ/CS/HA配制成終濃度10mg/ml復(fù)合仿生基質(zhì)溶膠,取3ml調(diào)至中性,加入EDAC20μl攪拌均勻;用1ml注射器吸入中性復(fù)合膠并注入軟骨缺損模型,放入37℃孵箱,6min后觀察成凝膠情況;設(shè)計(jì)軟骨微粒獲取裝置,包括主機(jī)、刨頭、踏板;利用裝置獲取軟骨微粒在100-120μm之間,符合最佳增殖粒徑標(biāo)準(zhǔn);COLⅡ/CS/HA按質(zhì)量比8:1.5:0.5溶于0.15mol/lHCL中,取3ml調(diào)至中性備用;軟骨微粒按5×104個(gè)/ml與COLⅡ/CS/HA溶膠混合,加入EDAC20μl攪拌均勻,注入軟骨缺損模型中,升溫6min成凝膠后,放入高糖培養(yǎng)基中培養(yǎng),1周后進(jìn)行大體,免疫熒光和組織學(xué)觀察。結(jié)果:制備的軟骨微粒即刻顯微鏡觀察軟骨細(xì)胞分布均勻,呈強(qiáng)折光性,可見部分軟骨陷窩被切開;培養(yǎng)1d各組微粒均有少量細(xì)胞脫落;3d時(shí)可見較多軟骨細(xì)胞從軟骨微粒中脫落、增殖;7d時(shí)大量軟骨細(xì)胞脫落、增殖,幾乎長(zhǎng)滿板底,貼壁并呈長(zhǎng)條狀。在3個(gè)時(shí)間點(diǎn)對(duì)各組軟骨細(xì)胞脫落、增殖情況計(jì)數(shù),A組較B、C、D組軟骨細(xì)胞增殖好,差異均有統(tǒng)計(jì)學(xué)意義(P0.05),最佳增殖粒徑為小于120μm的軟骨微粒;制備的仿生基質(zhì)溶膠3ml,加入EDAC20μl交聯(lián)劑后低溫下呈溶膠狀,具有流動(dòng)性,升溫一定時(shí)間后,變成凝膠狀,彈性、強(qiáng)度良好;軟骨微粒復(fù)合仿生基質(zhì)溶膠體外共培養(yǎng)1W,大體觀察見:外觀呈淡紅色,培養(yǎng)基充分浸入基質(zhì)中,觸之表面光滑并具有一定的彈性和強(qiáng)度;免疫熒光示:軟骨微粒中的軟骨細(xì)胞及脫落、增殖的軟骨細(xì)胞成活良好;HE染色示:軟骨微粒均勻分散于凝膠中,軟骨微粒中軟骨細(xì)胞存活良好;阿爾新蘭染色示:軟骨細(xì)胞從微粒中脫落、增殖并擴(kuò)散至周圍基質(zhì)中;天狼猩紅染色偏振光示:軟骨微粒與凝膠基質(zhì)融合良好,且成份基本相同。結(jié)論:軟骨微粒體外培養(yǎng)完全可以存活,不同粒徑的軟骨微粒均有軟骨細(xì)胞脫落、增殖。證實(shí)粒徑小于120μm增殖效果最好,粒徑越小切開軟骨陷窩的機(jī)率大,細(xì)胞從微粒中脫落就多,此方法獲取的軟骨微粒作為組織工程軟骨種子細(xì)胞來源具有可行性;COLⅡ/CS/HA仿生基質(zhì)溶膠加入EDAC交聯(lián)劑后利用溫控相變?cè)?6min能夠由溶膠成凝膠,且凝膠彈性、強(qiáng)度好,具備植入體內(nèi)缺損處的初始穩(wěn)定性,滿足作為支架材料要求;軟骨微粒復(fù)合仿生基質(zhì)溶膠體外共培養(yǎng),混合物中有大量軟骨細(xì)胞脫落、增殖,軟骨微粒與基質(zhì)融合較好,體外成軟骨具有可行性,輔以最佳的在體培養(yǎng)增殖環(huán)境,能形成透明軟骨,是修復(fù)關(guān)節(jié)軟骨缺損一種新方法。
[Abstract]:Background: the clinical incidence of articular cartilage injury is becoming more and more high and is being paid great attention to. Because normal hyaline cartilage is a special tissue, no blood vessel, nerve, its self repair ability is low and the cartilage defect with diameter more than 4mm can not be repaired completely. After the injury of cartilage, it can cause joint swelling and pain and affect motor function. The traditional methods for the treatment of articular cartilage defects are: micro fracture technique, autologous / allograft osteochondral transplantation, but there are some defects and shortcomings. With the development of tissue engineering, autologous chondrocyte transplantation (ACI) technology is used in clinical application, and a significant treatment effect has been obtained. However, there are still obvious defects: first, the culture of cartilage cells in vitro, amplification, is difficult to simulate in the body microenvironment, the lack of the monitoring of the body's immune system, the implanted cartilage cells usually have phenotypic changes, and the final formed tissues are essentially different from the natural cartilage in both the structure and the composition, followed by the method. The two operation was implanted to repair the defect, the duration of the treatment was long, the body damage was large, and the patient had great physical pain and economic burden. Objective: to construct tissue engineering cartilage in vitro by tissue engineering technique, take the cartilage of knee joint of Guizhou small Xiang pig under aseptic condition, make cartilage microparticles, and observe the medium soft cartilage after culture in vitro. Bone cells shedding, proliferation, screening the best size of the proliferation effect, and then using the best cartilage particles as seed cells; preparation of type II collagen, chondroitin sulfate, hyaluronic acid (COL II /CS/HA) bionic matrix gel, adding EDAC crosslinker, after heating the gel to detect the gelation performance; cartilage microparticles and imitation The feasibility of construction of tissue engineered cartilage in vitro was evaluated through gross and histological examination. After the success of this experiment, we hope to provide a basis for a surgical treatment of articular cartilage defects. Methods: the knee joint of 3-5 months old Guizhou miniature pigs was taken to make the cartilage microparticle shape, and the pore size was 120 mu m, respectively. 212 micron m, 475 m, 880 micron particle sieves were filtered, and the cartilage particles of different sizes were collected. According to the size of A, B, C, D groups, each group was given the same quality, and 3 12 hole culture plates were planted evenly. The 1 plates were counted for 1D, 3D and 7d respectively. The diameter of 8mm full thickness cartilage defect was prepared on the knee joint of fresh pig. COL II /CS/HA was prepared into a final concentration 10mg/ml composite biomimetic matrix sol, 3ml to neutral, and EDAC20 Mu l to be stirred, and neutral compound glue was inhaled with 1ml syringe and injected into the cartilage defect model, and the gel condition was observed at 37 C, and the gel condition was observed after 6min. A cartilage microparticle acquisition device, including a host, a planer, and a pedal, is used to obtain the cartilage particles between 100-120 mu m and the optimum diameter standard. COL II /CS/HA is dissolved in 0.15mol/lHCL by mass ratio 8:1.5:0.5 to neutral reserve, and the cartilage particles are mixed by 5 x 104 /ml with COL II /CS/HA sols, adding EDAC20 Mu L and even mixing, In the model of injecting cartilage defect, after heating 6min into gelatin, it was cultured in high glucose medium. After 1 weeks, gross, immunofluorescence and histological observation were carried out. Results: the cartilage microscopes were prepared by the immediate microscope to observe the uniform distribution of cartilage cells, strong refraction, and some cartilage lacunae were cut, and a small amount of cells were removed from each group of 1D. At 3D, many chondrocytes were found to fall off from the cartilage particles and proliferate. When 7d, a large number of chondrocytes were dropped, proliferated, almost full of plate and long strip. At 3 time points, the chondrocytes were dropped, the proliferation was counted, and the proliferation of chondrocytes in group A was better than that in B, C and D group, the difference was statistically significant (P0.05), and the best proliferation particle size was the same. The cartilage particles less than 120 mu m; the prepared biomimetic matrix sol 3ml, adding EDAC20 Mu l crosslinking agent, is gelatinous and fluidity at low temperature. After a certain time heating, it becomes gelatinous, elastic and strong, and the cartilage microparticles compound bionic matrix sol is co cultured in vitro for 1W. The general observation is that the appearance is light red, the medium is fully immersed in the matrix. The surface of the contact is smooth and has a certain elasticity and strength; immunofluorescence shows that the cartilage cells in the cartilage particles and the chondrocytes proliferate well; HE staining shows that the cartilage particles are evenly dispersed in the gel, and the cartilage cells in the cartilage microparticles are well alive; alnew blue shows that the cartilage cells fall off the particles and proliferate. Diffused into the surrounding matrix, Sirius red staining polarized light showed that the cartilage particles were well fused with the gel matrix, and the composition was basically the same. Conclusion: the cartilage microparticles can survive in vitro, and the cartilage particles with different particle sizes have the cartilage cells falling off and proliferating. It is proved that the particle size is smaller than 120 m, the smaller the particle size is, the smaller the diameter of the cartilage is cut in the cartilage. The probability of the nests is large and the cells fall off from the particles. The cartilage particles obtained by this method are feasible as the source of the tissue engineering cartilage seed cells. After adding EDAC crosslinker to the COL II /CS/HA bionic matrix sol, the 6min can be formed by the sol gel, and the gel is elastic and has good strength and has the defects in the body. The initial stability is satisfied as the requirement of scaffold material; cartilage microparticle composite biomimetic matrix sol is co cultured in vitro. A large number of chondrocytes in the mixture fall off, proliferate, the fusion of cartilage particles and matrix is better, and the cartilage is feasible in vitro, supplemented with the best in body culture environment, can form hyaline cartilage, is the repair of articular cartilage deficiency. Undermine a new method.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R684.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 寧志剛;楊柳;;膠原蛋白水凝膠在軟骨組織工程中的應(yīng)用[J];中國(guó)骨傷;2011年10期

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本文編號(hào)：2056663