骨軟骨脫細(xì)胞基質(zhì)材料研制及修復(fù)功能動(dòng)物實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-09-11 12:36
【摘要】：研究背景: 關(guān)節(jié)骨軟骨由軟骨,軟骨下骨及之間的連接結(jié)構(gòu)構(gòu)成。人體關(guān)節(jié)活動(dòng)使用量巨大,極容易在創(chuàng)傷、腫瘤及急慢性炎癥中導(dǎo)致關(guān)節(jié)軟骨或骨軟骨損傷。軟骨損傷也常會(huì)發(fā)展致滑膜、關(guān)節(jié)囊及軟骨下骨,導(dǎo)致骨軟骨損傷,骨軟骨損傷通常伴隨關(guān)節(jié)機(jī)械應(yīng)力改變,如不治療會(huì)進(jìn)一步導(dǎo)致退行性關(guān)節(jié)炎發(fā)生,患者功能障礙及生活質(zhì)量下降。關(guān)節(jié)軟骨無血液、淋巴及神經(jīng),僅包含單一軟骨細(xì)胞,細(xì)胞外基質(zhì)細(xì)胞比高并且缺少局部祖細(xì)胞,關(guān)節(jié)軟骨自身修復(fù)能力很差。關(guān)節(jié)軟骨下骨主要對(duì)關(guān)節(jié)軟骨起支撐作用,軟骨下骨損傷通常采用替代物植入治療,替代物的血管化不足將不能及時(shí)修復(fù),大塊骨缺損將明顯影響其支撐作用。骨軟骨之間的連接結(jié)構(gòu)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(jié)構(gòu)微細(xì)并復(fù)雜,骨軟骨連接區(qū)損傷可能會(huì)影響其重要的生理功能。關(guān)節(jié)軟骨及骨軟骨損傷自身修復(fù)困難,關(guān)節(jié)骨軟骨損傷治療成為目前骨關(guān)節(jié)外科難題之一。 對(duì)軟骨及骨軟骨缺損修復(fù)方法較多,較有效的方法包括采用具備軟骨、軟骨下骨及其間的連接結(jié)構(gòu)的自體及異體骨軟骨移植;而僅對(duì)軟骨的修復(fù)較具潛力的方法主要是組織工程技術(shù),其中MACI(基質(zhì)介導(dǎo)自體軟骨細(xì)胞移植)技術(shù)能達(dá)到部分透明軟骨修復(fù)效果,顯示較好的前景。MACI對(duì)骨軟骨損傷修復(fù)欠佳,而骨軟骨移植物來源有限,但MACI及骨軟骨技術(shù)提示帶軟骨、軟骨下骨及其間的連接結(jié)構(gòu)的骨軟骨單元移植并結(jié)合種子細(xì)胞可能是骨軟骨修復(fù)的潛在方案。理想的體外構(gòu)建的組織工程替代結(jié)構(gòu)應(yīng)該模擬關(guān)節(jié)組織的自然成分及結(jié)構(gòu)進(jìn)而恢復(fù)其正常功能,國內(nèi)外對(duì)組織工程骨軟骨復(fù)合組織的構(gòu)建研究,從“分層構(gòu)建”的可行性初探到“一體構(gòu)建”的動(dòng)物實(shí)驗(yàn)研究取得了階段性成果,然而目前尚存在缺損區(qū)修復(fù)組織質(zhì)量缺陷、與宿主界面整合欠佳及缺乏相應(yīng)力學(xué)功能等主要問題。由于骨及軟骨脫細(xì)胞在技術(shù)上取得一定進(jìn)展,通過粉碎軟骨并采用脫細(xì)胞試劑處理的方法可以將細(xì)胞成分去除,而保留軟骨細(xì)胞外基質(zhì)成分。 本課題擬在此基礎(chǔ)上,分別將含骨軟骨之間連接結(jié)構(gòu)的組織及軟骨進(jìn)行脫細(xì)胞,利用脫細(xì)胞基質(zhì)材料制備含生物來源骨軟骨連接結(jié)構(gòu)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連接結(jié)構(gòu),軟骨側(cè)構(gòu)建軟骨脫細(xì)胞基質(zhì)支架,保留脫細(xì)胞軟骨下骨,盡量模擬了骨軟骨結(jié)構(gòu)。通過體外接種骨髓來源間充質(zhì)干細(xì)胞構(gòu)建細(xì)胞-材料復(fù)合體,植入體內(nèi)修復(fù)動(dòng)物骨軟骨缺損模型并觀察軟骨生成效果;并且采用骨髓來源分離擴(kuò)增的內(nèi)皮祖細(xì)胞接種脫細(xì)胞骨基質(zhì)材料修復(fù)動(dòng)物大段骨缺損,通過血管化來促進(jìn)大段骨缺損的修復(fù),為研究軟骨下骨缺損修復(fù)提供新的方法。本研究將為仿生制備骨軟骨復(fù)合支架材料提供依據(jù),同時(shí)為構(gòu)建更為復(fù)雜的組織工程關(guān)節(jié)創(chuàng)造條件。在研究內(nèi)容上主要包括:(1)分別將軟骨粉、軟骨片及骨軟骨復(fù)合組織脫細(xì)胞處理并鑒定;(2)構(gòu)建含骨軟骨連接結(jié)構(gòu)的脫細(xì)胞骨軟骨復(fù)合組織工程支架;(3)利用骨髓間充質(zhì)干細(xì)胞復(fù)合含骨軟骨連接結(jié)構(gòu)的骨軟骨支架修復(fù)羊膝關(guān)節(jié)負(fù)重區(qū)骨軟骨缺損模型;(4)采用脫細(xì)胞骨基質(zhì)接種骨髓來源內(nèi)皮祖細(xì)胞修復(fù)兔尺骨大段骨缺損。 方法: 1.將天然人軟骨在蛋白酶抑制劑保護(hù)下粉碎,采用離心分選軟骨微粒,經(jīng)過軟骨脫細(xì)胞處理后制備軟骨脫細(xì)胞微粒懸液,行組織學(xué)及生化定量分析檢測(cè)。 2.制備直徑為8mm含骨軟骨連接結(jié)構(gòu)的骨軟骨組織塊,其軟骨側(cè)僅保留約100μm透明軟骨,經(jīng)脫細(xì)胞處理后予組織學(xué),生化定量分析檢測(cè)。采用凍干法及化學(xué)交聯(lián)法制備含骨軟骨連接結(jié)構(gòu)的脫細(xì)胞骨軟骨支架,培養(yǎng)羊骨髓間充質(zhì)干細(xì)胞,將羊骨髓間充質(zhì)干細(xì)胞接種于骨軟骨支架兩側(cè),體外培養(yǎng)7天行組織學(xué),組織相容性和細(xì)胞毒性檢測(cè)。 3.制備羊負(fù)重區(qū)骨軟骨缺損模型,分空白、植入空白支架及植入細(xì)胞支架復(fù)合物3組,3月后處死動(dòng)物取標(biāo)本行大體及組織學(xué)檢測(cè)。 4.構(gòu)建脫細(xì)胞骨支架及骨髓來源內(nèi)皮祖細(xì)胞復(fù)合物,制備兔尺骨缺損模型,分空白,脫細(xì)胞骨支架及細(xì)胞支架復(fù)合物3組修復(fù)兔尺骨缺損,2,4,8周處死動(dòng)物取標(biāo)本行組織學(xué)檢測(cè)。 結(jié)果: 1.組織學(xué)顯示直徑100μm以內(nèi)軟骨細(xì)胞微粒通過軟骨脫細(xì)胞處理后無細(xì)胞碎片殘留,甲苯胺藍(lán)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(zhì)結(jié)構(gòu)部分保留;生化定量結(jié)果表明DNA成分去除,保留大量細(xì)胞外基質(zhì)成分; 2.組織學(xué)顯示含部分透明軟骨的骨軟骨組織塊脫細(xì)胞處理后無細(xì)胞碎片殘留,軟骨側(cè)甲苯胺藍(lán)染色,番紅O及二型膠原免疫組化染色成陽性,骨軟骨連接結(jié)構(gòu)功能基本保留;生化定量結(jié)果表明DNA成分去除,保留大量細(xì)胞外基質(zhì)成分;含骨軟骨連接結(jié)構(gòu)的脫細(xì)胞骨軟骨支架經(jīng)組織學(xué)檢測(cè)骨軟骨支架連接完好,羊骨髓間充質(zhì)干細(xì)胞在支架上生長良好,軟骨側(cè)番紅O及二型膠原免疫染色陽性,電鏡及組織學(xué)顯示細(xì)胞生長良好,有細(xì)胞外基質(zhì)分泌; 3.修復(fù)羊負(fù)重區(qū)骨軟骨缺損模型實(shí)驗(yàn)結(jié)果顯示細(xì)胞支架復(fù)合修復(fù)組骨軟骨有較好修復(fù),空白支架組軟骨下骨基本修復(fù)、軟骨側(cè)無明顯修復(fù),空白對(duì)照組未見明顯修復(fù),缺損邊緣軟骨退變; 4.骨髓來源干細(xì)胞能通過體外分離擴(kuò)增,接種骨髓來源的內(nèi)皮祖細(xì)胞的脫細(xì)胞骨基質(zhì)支架體內(nèi)修復(fù)大段骨缺損后,修復(fù)組織內(nèi)微血管密度較空白支架組高(p0.05),并能對(duì)兔尺骨大段骨缺損進(jìn)行一定程度的修復(fù)。結(jié)論: 粉碎的軟骨微粒、厚度100μm左右軟骨片及僅含100μm厚透明軟骨的骨軟骨組織通過脫細(xì)胞處理,均可去除組織內(nèi)細(xì)胞成分,保留大部分細(xì)胞外基質(zhì)結(jié)構(gòu)功能;通過凍干及化學(xué)交聯(lián)可制備含骨軟骨連接結(jié)構(gòu)脫細(xì)胞骨軟骨支架,該支架軟骨側(cè)為軟骨脫細(xì)胞多孔支架,軟骨下骨側(cè)為脫細(xì)胞骨,骨軟骨連接結(jié)構(gòu)保留,該支架具備良好細(xì)胞相容性,無明顯細(xì)胞毒性;含骨軟骨連接結(jié)構(gòu)的脫細(xì)胞骨軟骨支架接種種子細(xì)胞能較好的修復(fù)羊負(fù)重區(qū)骨軟骨缺損;脫細(xì)胞骨支架及骨髓來源內(nèi)皮祖細(xì)胞復(fù)合物,對(duì)兔尺骨缺損修復(fù)及促進(jìn)微血管生成具備一定作用。
[Abstract]:Research background:
Articular cartilage is made up of cartilage, subchondral bone and connective structures. The use of large amounts of human joint activities is extremely easy to cause articular cartilage or osteochondral injury in trauma, tumor and acute or chronic inflammation. Cartilage injury often develops into synovium, articular capsule and subchondral bone, resulting in osteochondral injury, often accompanied by osteochondral injury. The articular cartilage has no blood, lymph and nerve, only a single chondrocyte, a high ratio of extracellular stromal cells and a lack of local progenitor cells. The articular cartilage has a poor ability to repair itself. Subchondral bone injuries are usually treated with substitutes implanted. Inadequate vascularization of substitutes will not be able to be repaired in time, and large bone defects will significantly affect the supporting effect. Bone-cartilage junctions include deep hyaline cartilage, tidal line, calcified cartilage, adhesive line and upper subchondral bone with fine structure. It is difficult for articular cartilage and osteochondral injury to repair itself. The treatment of articular cartilage injury has become one of the difficult problems in osteoarthroplasty.
There are many methods for repairing cartilage and osteochondral defects, including autogenous and allograft cartilage transplantation with cartilage, subchondral bone and its connective structure, while tissue engineering is the most promising method for cartilage repair only, in which MACI (matrix-mediated autologous chondrocyte transplantation) technique can achieve partial repair. MacI is not good for the repair of osteochondral injury, but the source of osteochondral grafts is limited. However, MACI and osteochondral techniques suggest that the transplantation of osteochondral units with cartilage, subchondral bone and its connective structures and the combination of seeding cells may be a potential option for osteochondral repair. The constructed tissue engineering substitution structure should simulate the natural composition and structure of joint tissue to restore its normal function. The construction of tissue engineering osteochondral composite tissue has been studied at home and abroad. From the feasibility of "layered construction" to the animal experimental study of "integrated construction", the results have been achieved at different stages. Due to the advances in bone and cartilage acellular technology, chondrocyte components can be removed by comminuting cartilage and using acellular reagents, while extracellular matrix components can be retained.
On this basis, the tissue and cartilage containing the connective structure between osteochondria were acellularized, and the osteochondral scaffolds containing biological osteochondral connective structure were prepared using acellular matrix materials. The scaffolds include deep hyaline cartilage, tidal line, calcified cartilage layer, adhesive line, subchondral bone plate and other physiological osteochondral connective materials. The bone marrow-derived mesenchymal stem cells (BMSCs) were inoculated in vitro to construct the cell-material complex and implanted in vivo to repair the animal osteochondral defect model and observe the effect of cartilage formation. Endothelial progenitor cells (EPCs) were seeded with acellular bone matrix (ABM) to repair large bone defects in animals, and vascularization was used to promote the repair of large bone defects. This study will provide a new method for the study of subchondral bone defect repair. Conditions include: (1) acellular treatment and identification of cartilage powder, cartilage slices and osteochondral composite tissue; (2) construction of acellular osteochondral composite tissue engineering scaffold with osteochondral junction; (3) repair of sheep knee joint negative by bone marrow mesenchymal stem cells and osteochondral composite scaffold with osteochondral junction structure. (4) Bone marrow-derived endothelial progenitor cells (EPCs) were seeded with acellular bone matrix to repair large ulnar bone defects in rabbits.
Method:
1. The natural human cartilage was crushed under the protection of protease inhibitor. The cartilage particles were centrifuged and separated. After the cartilage was acellular treated, the cartilage acellular particles suspension was prepared for histological and biochemical quantitative analysis.
2. The osteochondral tissue block with osteochondral junction structure of 8 mm in diameter was prepared, and only about 100 micron hyaline cartilage was retained on the cartilage side. After acellular treatment, histological and biochemical quantitative analysis were carried out. The acellular osteochondral scaffold containing osteochondral junction structure was prepared by freeze-drying method and chemical cross-linking method. Bone marrow mesenchymal stem cells were seeded on both sides of osteochondral scaffolds and cultured in vitro for 7 days for histological, histocompatibility and cytotoxicity tests.
3. Prepare sheep osteochondral defect model in load-bearing area, and divide into three groups: blank, blank and cellular scaffold composite. Samples were sacrificed 3 months later for gross and histological examination.
4. Acellular bone scaffold and bone marrow-derived endothelial progenitor cell complex were constructed to prepare rabbit ulna defect model. The rabbit ulna defect was repaired by three groups: blank group, acellular bone scaffold group and cell scaffold complex group. The rabbits were sacrificed at 2,4,8 weeks for histological examination.
Result:
1. Histological examination showed that chondrocyte particles with diameter less than 100 micron were free of cell debris after acellular treatment, toluidine blue staining, Safranine O and collagen II immunohistochemical staining were positive, and the structure of cartilage matrix was partly preserved under light microscope.
2. Histology showed that there was no residual cell fragments, toluidine blue staining on cartilage side, Safranine O and collagen II immunohistochemical staining were positive, and the structure and function of osteochondral junction were basically preserved after acellular treatment of osteochondral tissue with hyaline cartilage. The acellular osteochondral scaffold with cartilage junction structure was well-connected by histological examination. The goat bone marrow mesenchymal stem cells grew well on the scaffold. Safranine O and collagen II immunostaining were positive on the cartilage side. Electron microscopy and histology showed that the cells grew well and secreted extracellular matrix.
3. The experimental results of repairing sheep's osteochondral defects in the load-bearing area showed that the osteochondral defects were repaired well in the cell scaffold group, and the subchondral bones were basically repaired in the blank scaffold group, and the cartilage side was not repaired obviously in the blank control group.
4. Bone marrow-derived stem cells can be isolated and amplified in vitro, and the acellular bone matrix scaffolds inoculated with bone marrow-derived endothelial progenitor cells can repair large bone defects in vivo. The microvessel density in the repaired tissues is higher than that in the blank scaffolds group (p0.05), and can repair large bone defects of rabbit ulna to a certain extent.
By acellular treatment, the chondrocartilage with a thickness of about 100 microns, and the osteochondral tissue with only 100 microns of hyaline cartilage can remove the intracellular components and retain most of the structure and function of extracellular matrix. The scaffolds have good cell compatibility and no obvious cytotoxicity. The seeding cells of acellular osteochondral scaffolds with osteochondral junction can repair the bone and cartilage defects of sheep in the load-bearing area. Endothelial progenitor cell complex can play an important role in repairing ulnar defects and promoting angiogenesis in rabbits.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
本文編號(hào)：2236708
[Abstract]:Research background:
Articular cartilage is made up of cartilage, subchondral bone and connective structures. The use of large amounts of human joint activities is extremely easy to cause articular cartilage or osteochondral injury in trauma, tumor and acute or chronic inflammation. Cartilage injury often develops into synovium, articular capsule and subchondral bone, resulting in osteochondral injury, often accompanied by osteochondral injury. The articular cartilage has no blood, lymph and nerve, only a single chondrocyte, a high ratio of extracellular stromal cells and a lack of local progenitor cells. The articular cartilage has a poor ability to repair itself. Subchondral bone injuries are usually treated with substitutes implanted. Inadequate vascularization of substitutes will not be able to be repaired in time, and large bone defects will significantly affect the supporting effect. Bone-cartilage junctions include deep hyaline cartilage, tidal line, calcified cartilage, adhesive line and upper subchondral bone with fine structure. It is difficult for articular cartilage and osteochondral injury to repair itself. The treatment of articular cartilage injury has become one of the difficult problems in osteoarthroplasty.
There are many methods for repairing cartilage and osteochondral defects, including autogenous and allograft cartilage transplantation with cartilage, subchondral bone and its connective structure, while tissue engineering is the most promising method for cartilage repair only, in which MACI (matrix-mediated autologous chondrocyte transplantation) technique can achieve partial repair. MacI is not good for the repair of osteochondral injury, but the source of osteochondral grafts is limited. However, MACI and osteochondral techniques suggest that the transplantation of osteochondral units with cartilage, subchondral bone and its connective structures and the combination of seeding cells may be a potential option for osteochondral repair. The constructed tissue engineering substitution structure should simulate the natural composition and structure of joint tissue to restore its normal function. The construction of tissue engineering osteochondral composite tissue has been studied at home and abroad. From the feasibility of "layered construction" to the animal experimental study of "integrated construction", the results have been achieved at different stages. Due to the advances in bone and cartilage acellular technology, chondrocyte components can be removed by comminuting cartilage and using acellular reagents, while extracellular matrix components can be retained.
On this basis, the tissue and cartilage containing the connective structure between osteochondria were acellularized, and the osteochondral scaffolds containing biological osteochondral connective structure were prepared using acellular matrix materials. The scaffolds include deep hyaline cartilage, tidal line, calcified cartilage layer, adhesive line, subchondral bone plate and other physiological osteochondral connective materials. The bone marrow-derived mesenchymal stem cells (BMSCs) were inoculated in vitro to construct the cell-material complex and implanted in vivo to repair the animal osteochondral defect model and observe the effect of cartilage formation. Endothelial progenitor cells (EPCs) were seeded with acellular bone matrix (ABM) to repair large bone defects in animals, and vascularization was used to promote the repair of large bone defects. This study will provide a new method for the study of subchondral bone defect repair. Conditions include: (1) acellular treatment and identification of cartilage powder, cartilage slices and osteochondral composite tissue; (2) construction of acellular osteochondral composite tissue engineering scaffold with osteochondral junction; (3) repair of sheep knee joint negative by bone marrow mesenchymal stem cells and osteochondral composite scaffold with osteochondral junction structure. (4) Bone marrow-derived endothelial progenitor cells (EPCs) were seeded with acellular bone matrix to repair large ulnar bone defects in rabbits.
Method:
1. The natural human cartilage was crushed under the protection of protease inhibitor. The cartilage particles were centrifuged and separated. After the cartilage was acellular treated, the cartilage acellular particles suspension was prepared for histological and biochemical quantitative analysis.
2. The osteochondral tissue block with osteochondral junction structure of 8 mm in diameter was prepared, and only about 100 micron hyaline cartilage was retained on the cartilage side. After acellular treatment, histological and biochemical quantitative analysis were carried out. The acellular osteochondral scaffold containing osteochondral junction structure was prepared by freeze-drying method and chemical cross-linking method. Bone marrow mesenchymal stem cells were seeded on both sides of osteochondral scaffolds and cultured in vitro for 7 days for histological, histocompatibility and cytotoxicity tests.
3. Prepare sheep osteochondral defect model in load-bearing area, and divide into three groups: blank, blank and cellular scaffold composite. Samples were sacrificed 3 months later for gross and histological examination.
4. Acellular bone scaffold and bone marrow-derived endothelial progenitor cell complex were constructed to prepare rabbit ulna defect model. The rabbit ulna defect was repaired by three groups: blank group, acellular bone scaffold group and cell scaffold complex group. The rabbits were sacrificed at 2,4,8 weeks for histological examination.
Result:
1. Histological examination showed that chondrocyte particles with diameter less than 100 micron were free of cell debris after acellular treatment, toluidine blue staining, Safranine O and collagen II immunohistochemical staining were positive, and the structure of cartilage matrix was partly preserved under light microscope.
2. Histology showed that there was no residual cell fragments, toluidine blue staining on cartilage side, Safranine O and collagen II immunohistochemical staining were positive, and the structure and function of osteochondral junction were basically preserved after acellular treatment of osteochondral tissue with hyaline cartilage. The acellular osteochondral scaffold with cartilage junction structure was well-connected by histological examination. The goat bone marrow mesenchymal stem cells grew well on the scaffold. Safranine O and collagen II immunostaining were positive on the cartilage side. Electron microscopy and histology showed that the cells grew well and secreted extracellular matrix.
3. The experimental results of repairing sheep's osteochondral defects in the load-bearing area showed that the osteochondral defects were repaired well in the cell scaffold group, and the subchondral bones were basically repaired in the blank scaffold group, and the cartilage side was not repaired obviously in the blank control group.
4. Bone marrow-derived stem cells can be isolated and amplified in vitro, and the acellular bone matrix scaffolds inoculated with bone marrow-derived endothelial progenitor cells can repair large bone defects in vivo. The microvessel density in the repaired tissues is higher than that in the blank scaffolds group (p0.05), and can repair large bone defects of rabbit ulna to a certain extent.
By acellular treatment, the chondrocartilage with a thickness of about 100 microns, and the osteochondral tissue with only 100 microns of hyaline cartilage can remove the intracellular components and retain most of the structure and function of extracellular matrix. The scaffolds have good cell compatibility and no obvious cytotoxicity. The seeding cells of acellular osteochondral scaffolds with osteochondral junction can repair the bone and cartilage defects of sheep in the load-bearing area. Endothelial progenitor cell complex can play an important role in repairing ulnar defects and promoting angiogenesis in rabbits.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
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