【摘要】：背景及目的: 人體關節(jié)活動量巨大,極容易在創(chuàng)傷、腫瘤及急慢性炎癥中導致關節(jié)骨軟骨損傷。骨軟骨損傷通常伴隨關節(jié)機械應力改變,如不及時治療會進一步導致退行性骨關節(jié)炎發(fā)生,大多軟骨損傷也會發(fā)展至滑膜、關節(jié)囊及軟骨下骨,導致更嚴重的骨軟骨損傷,患肢功能障礙及生活質量下降。關節(jié)骨軟骨由軟骨,軟骨下骨及之間的鈣化層構成。關節(jié)軟骨基本無血液、淋巴及神經(jīng),僅包含單一軟骨細胞,細胞外基質細胞比高并且缺少局部祖細胞,導致關節(jié)軟骨自身修復能力很差。關節(jié)軟骨下骨主要對關節(jié)軟骨起支撐作用,軟骨下骨替代物的血管化不足將導致其不能及時修復,大塊軟骨下骨缺損將明顯影響其支撐作用。骨軟骨之間的鈣化層包含深層透明軟骨、潮線、鈣化軟骨、粘合線及上層軟骨下骨,其結構微細并復雜,難于體外構建,但骨軟骨連接區(qū)具備重要的生理功能。 目前臨床對軟骨的修復較好方法主要是MACI技術,能達到部分透明軟骨修復效果;對骨軟骨損傷修復的主要方法包括自體及異體骨軟骨移植,其具備軟骨、軟骨下骨及其間的鈣化層,能取得一定臨床效果。但是MACI對骨軟骨損傷修復欠佳,而骨軟骨移植物來源有限,但MACI及骨軟骨技術提示帶軟骨、軟骨下骨及其間的鈣化層的骨軟骨單元移植并結合種子細胞可能是骨軟骨修復的潛在方案。理想的體外構建的組織工程替代結構應該模擬關節(jié)組織的自然結構進而恢復其正常功能,目前的骨軟骨組織工程構建方法難以模擬包含骨軟骨鈣化層在內的骨軟骨組織。由于骨及軟骨脫細胞在技術上取得一定進展,通過粉碎軟骨基質的方法可以將組織細胞成分去除,而保留軟骨細胞外基質成分。本課題擬在此基礎上,分別將含骨軟骨之間鈣化層的組織及軟骨進行脫細胞,利用脫細胞基質材料制備含生物來源骨軟骨鈣化層的骨軟骨支架材料,該支架包含深層透明軟骨、潮線、鈣化軟骨層、粘合線、軟骨下骨板等生理骨軟骨鈣化層,骨軟骨上方交聯(lián)多孔軟骨脫細胞基質支架并體外接種骨髓來源間充質干細胞構建細胞-材料復合體,觀察細胞生物學改變。本研究將為仿生制備骨軟骨復合支架材料提供依據(jù),同時為構建更為復雜的組織工程關節(jié)創(chuàng)造條件。 本研究主要內容包括:(1)分別將軟骨粉、軟骨片及骨軟骨復合組織脫細胞處理,鑒定脫細胞程度并檢驗脫細胞對骨軟骨鈣化層功能的影響;(2)構建含骨軟骨鈣化層的脫細胞骨軟骨復合組織工程支架,鑒定骨軟骨支架并體外檢測支架生物相容性。 方法: 1.將天然羊軟骨在蛋白酶抑制劑保護下粉碎,采用離心分選軟骨微粒,經(jīng)過軟骨脫細胞處理后制備軟骨脫細胞微粒懸液,檢測其組織學,生化定量分析及理化性能檢測。 2.制備直徑為8mm含骨軟骨鈣化層的骨軟骨組織塊,其軟骨側僅保留約100um透明軟骨,經(jīng)脫細胞處理后予組織學,生物化學及理化性能檢測。 3.采用凍干法及化學交聯(lián)法制備含骨軟骨鈣化層的脫細胞骨軟骨支架,予組織學、生物化學及理化性能檢測,同時將擴增培養(yǎng)的羊骨髓間充質干細胞接種于含鈣化層骨軟骨支架兩側,體外培養(yǎng)7天行組織相容性和細胞毒性檢測。 結果: 1.組織學顯示直徑100um以內軟骨細胞微粒通過軟骨脫細胞處理后無細胞碎片殘留,甲苯胺藍染色,番紅O及二型膠原免疫組化染色成陽性,光鏡下見軟骨基質結構部分保留;生化定量結果表明DNA成分去除,保留大量細胞外基質成分; 2.組織學顯示含部分透明軟骨的骨軟骨組織塊脫細胞處理后無細胞碎片殘留,軟骨側甲苯胺藍染色,番紅O及二型膠原免疫組化染色成陽性,骨軟骨鈣化層功能基本保留;生化定量結果表明DNA成分去除,保留大量細胞外基質成分; 3.含骨軟骨鈣化層的脫細胞骨軟骨支架經(jīng)組織學檢測骨軟骨支架連接完好,羊骨髓間充質干細胞在支架上生長良好,電鏡及組織學顯示細胞生長良好,有細胞外基質分泌。 結論: 1.直徑100um內軟骨組織可以通過脫細胞處理去除細胞結構并保留大量細胞外基質成分;2.僅含100um厚透明軟骨的骨軟骨組織通過脫細胞處理,可去除組織內細胞成分,并保留骨軟骨間鈣化層;3.通過凍干及化學交聯(lián)可制備含骨軟骨鈣化層脫細胞骨軟骨支架,該支架軟骨側為軟骨脫細胞多孔支架,軟骨下骨側為脫細胞骨,骨軟骨鈣化層保留,該支架具備良好細胞相容性,無明顯細胞毒性。
[Abstract]:Background and purpose: The joint activity of the human body is huge, and the joint bone cartilage can be easily caused in the wound, the tumor and the acute and chronic inflammation Injury. The damage of the bone cartilage is usually accompanied by a change in the mechanical stress of the joint, such as the non-timely treatment can further lead to the occurrence of degenerative osteoarthritis, and most of the cartilage damage can also be developed to the synovium, the joint capsule and the subchondral bone, resulting in more severe bone-cartilage damage, limb dysfunction and quality of life. The articular cartilage is composed of the cartilage, the subchondral bone and the calcified layer between the subchondral bone and the cartilage. The articular cartilage is basically free of blood, lymph and nerves, only contains a single cartilage cell, the extracellular matrix cell ratio is high and the local progenitor cells are absent, which leads to the self-healing capacity of the articular cartilage itself. The articular cartilage subchondral bone is mainly supported by the articular cartilage, and the hypovascularisation of the subchondral bone substitute will cause it to not be repaired in time, and the subchondral bone defect will significantly affect its support The calcified layer between the bone and cartilage comprises the deep transparent cartilage, the tidal line, the calcified cartilage, the adhesive line and the upper layer of the subchondral bone, the structure is fine and complex, it is difficult to construct in vitro, but the joint area of the bone cartilage has an important physiological function. The method for repairing the cartilage is mainly based on the MACI technique, which can achieve the effect of partial transparent cartilage repair, and the main method for repairing the bone cartilage injury comprises the autograft and the allogenic bone cartilage graft, which is provided with the cartilage, the subchondral bone and the calcified layer between the cartilage and the subchondral bone, The clinical effect is fixed. However, the repair of the bone cartilage damage is not good, but the source of the bone cartilage graft is limited, but the MACI and the bone cartilage technique suggest that the bone cartilage unit with the cartilage, the subchondral bone and the calcified layer between the cartilage, the subchondral bone and the calcified layer between the cartilage and the subchondral bone may be a bone cartilage repair. The alternative structure of the ideal in vitro construction of the tissue engineering should mimic the natural structure of the joint tissue and restore its normal function, and the present construction method of the bone cartilage tissue engineering is difficult to simulate the calcified layer containing the bone cartilage. The bone and cartilage tissue. Due to the fact that bone and cartilage decells have made some progress in the art, the tissue cell components can be removed by the method of crushing the cartilage matrix, while the chondrocytes are retained. The subject is to take the tissue and the cartilage of the calcified layer between the bone-containing cartilage and the cartilage to decell, and use the acellular matrix material to prepare the bone cartilage support material containing the biological origin bone cartilage calcification layer, which comprises the deep transparent cartilage, the tide line and the calcified cartilage. a cell-material complex is constructed by a layer, an adhesive line, a cartilage subchondral bone plate, a physiological bone cartilage calcification layer, a cross-linked porous cartilage acellular matrix scaffold above the bone cartilage, and the bone marrow-derived mesenchymal stem cells are inoculated in vitro to construct a cell-material complex, Biological changes. The study will provide the basis for biomimetic preparation of the bone-cartilage composite scaffold material, and at the same time to construct a more complex tissue engineering closure The main contents of this study are as follows: (1) the cartilage powder, the cartilage sheet and the bone cartilage composite tissue are respectively decell-treated, the degree of decell is identified, and the acellular-to-bone cartilage is tested. the effect of the function of the calcified layer; (2) the scaffold of the acellular bone cartilage composite tissue engineering scaffold containing the bone cartilage calcification layer is constructed, the bone cartilage scaffold is identified and the in vitro test is carried out, test support The method comprises the following steps of:1, the natural sheep cartilage is crushed under the protection of a protease inhibitor, 2. Preparation of a bone-cartilage tissue mass with a diameter of 8 mm with a bone-cartilage-calcified layer, whose cartilage side only retains about 100 um of the transparent cartilage, and the cartilage side only retains about 100 um of the transparent cartilage, and the cartilage side only retains about 100 um of the transparent cartilage, and the cartilage side only retains about 100 um of the transparent cartilage, and The histological, biochemical and physical and chemical properties of the bone-containing cartilage-containing layer were prepared by freeze-drying and chemical cross-linking. The bone marrow of the cultured sheep was also detected by the methods of histology, biochemistry and physical and chemical properties. Mesenchymal stem cells are seeded on both sides of the calcified layer of bone cartilage support in vitro The histocompatibility and cytotoxicity of 7 day lines were tested. The results were as follows:1. There were no cell debris left, toluidine blue staining, Panyu O and type 2 after the treatment of the chondrocyte with the diameter of 100 um. The immunohistochemical staining of the collagen was positive, and the structure of the cartilage matrix was retained under the light microscope. The results showed that the DNA component was removed and a large amount of extracellular matrix components were retained. Cartilage-side methamidon-blue staining, Panyu-O and 2-type collagen immunohistochemical staining were positive, and the function of the bone-cartilage calcification layer was basic. the biochemical quantitative results indicate that the DNA component is removed and a large amount of the extracellular matrix component is retained; and the acellular bone cartilage scaffold containing the bone cartilage calcification layer is histologically detected, the bone cartilage scaffold is connected in good condition, and the bone marrow mesenchymal stem cells of the sheep in a stand The growth of the cells was good, and the electron microscope and the histology showed that the cells were well-grown and the extracellular matrix was secreted. in that 00 um, the cartilage tissue can remove the cell structure by decell treatment and retain a large amount of extracellular matrix component;2. only 100 um thick transparent soft tissue the bone cartilage tissue of the bone can remove the cell components in the tissue by decell treatment, and the calcification layer between the bone cartilage can be retained; and 3. the bone-containing cartilage-containing calcified layer acellular bone cartilage scaffold can be prepared by freeze-drying and chemical cross-linking, wherein the cartilage side of the scaffold is a cartilage acellular porous scaffold, a cartilage,
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R314

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本文編號：2508860