組織工程纖維環(huán)—髓核雙相支架的制備與性能評(píng)估
發(fā)布時(shí)間：2018-07-31 05:40
【摘要】：目的：以天然豬骨脫鈣基質(zhì)明膠(BMG,主要成分含Ⅰ型膠原)和軟骨細(xì)胞外基質(zhì)(CECM,主要成分為Ⅱ型膠原/GAG)為材料,利用仿生學(xué)原理研制新型纖維環(huán)-髓核雙相支架,并探討其作為組織工程椎間盤(pán)支架的可行性。 方法： 1.纖維環(huán)支架：取豬股骨近端松質(zhì)骨,環(huán)鉆鉆取外徑10mm、內(nèi)徑5mm、厚3mm的中空環(huán)狀骨環(huán),高壓水沖洗,依次進(jìn)行脫脂、脫鈣、脫細(xì)胞等處理,制成中空環(huán)狀脫鈣骨基質(zhì)明膠為材料的椎間盤(pán)纖維環(huán)支架。評(píng)估支架材料的孔徑和孔隙率、吸水率,采用微型力學(xué)測(cè)試機(jī)行力學(xué)測(cè)試,采用分離培養(yǎng)的兔脂肪干細(xì)胞和MTT法分析支架浸提液毒性。 2.纖維環(huán)-髓核雙相支架：將豬關(guān)節(jié)軟骨粉碎采用梯度離心法選取5μm～100μm軟骨微絲,脫細(xì)胞等處理后制備成質(zhì)量體積比為3.5%的懸液,將軟骨漿料懸液注入中空環(huán)狀脫鈣骨基質(zhì)纖維環(huán)支架中央冷凍凍干,采用紫外線(xiàn)以及化學(xué)交聯(lián)劑碳化二亞胺等對(duì)支架進(jìn)行交聯(lián)。行組織學(xué)及掃描電鏡觀察,測(cè)定支架孔徑和孔隙率、吸水率。將兔脂肪干細(xì)胞種植在支架上,倒置顯微鏡以及掃描電鏡觀察細(xì)胞在支架上的粘附以及生長(zhǎng)情況。 3.雙相纖維環(huán)支架：取豬掌骨中央圓形區(qū)域,鋸取厚5mm的皮質(zhì)骨-松質(zhì)骨環(huán),沖洗、脫鈣、脫細(xì)胞制成皮質(zhì)骨-松質(zhì)骨環(huán)雙相纖維環(huán)支架。觀察支架的孔徑及孔隙,測(cè)定吸水率及孔隙率,用微型力學(xué)測(cè)試機(jī)行力學(xué)測(cè)試。結(jié)果： 1.纖維環(huán)支架：以松質(zhì)骨脫鈣骨基質(zhì)明膠制備的纖維環(huán)支架為乳白色的環(huán)形多孔結(jié)構(gòu),孔徑大致均勻,孔隙相通。濕性狀態(tài)下質(zhì)地柔軟。HE染色顯示組織內(nèi)無(wú)細(xì)胞結(jié)構(gòu)殘留。光鏡及掃描電鏡示支架孔隙均勻,支架纖維環(huán)部孔徑為401.44±13.1μm,孔隙率為62.12±1.52%,吸水率為409.774±11.34%。生物力學(xué)結(jié)果示支架的彈性模量為47.75±6.32MPa。細(xì)胞增殖曲線(xiàn)顯示不同濃度支架浸提液與對(duì)照DMEM培養(yǎng)液吸光度值比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 2.纖維環(huán)-髓核雙相支架：以松質(zhì)骨脫鈣骨基質(zhì)明膠與軟骨細(xì)胞外基質(zhì)構(gòu)建的纖維環(huán)-髓核雙相支架為淡黃白色的圓形多孔結(jié)構(gòu),孔隙相通,濕性狀態(tài)下質(zhì)地柔軟�？讖酱笾戮鶆�,纖維環(huán)部孔徑大于髓核部,支架髓核部孔徑為101.3±22.5μm,孔隙率為83.24±4.31%,吸水率為421.45±8.43%,支架總體彈性模量為53.45±3.21MPa。HE染色顯示組織內(nèi)無(wú)細(xì)胞結(jié)構(gòu)殘留。光鏡及掃描電鏡示支架孔隙均勻。倒置顯微鏡下細(xì)胞在支架上粘附良好。掃描電鏡下細(xì)胞在支架上有良好的粘附,分布均勻,生長(zhǎng)良好。 3.纖維環(huán)雙相支架：以皮質(zhì)骨-松質(zhì)骨環(huán)制備的的纖維環(huán)雙相支架為淡黃色類(lèi)圓形多孔結(jié)構(gòu),孔徑、孔隙率以及吸水率比松質(zhì)骨纖維環(huán)支架小,孔隙率為51.41±2.71%,吸水率為350.15±3.21%。 結(jié)論： 1.以松質(zhì)骨脫鈣骨基質(zhì)明膠制備的纖維環(huán)支架具有良好的孔隙率、孔徑、吸水率、生物相容性,彈性模量與纖維環(huán)在同一數(shù)量級(jí)。 2.以松質(zhì)骨脫鈣基質(zhì)明膠和軟骨細(xì)胞外基質(zhì)制備的纖維環(huán)-髓核雙相支架具有良好的孔徑、孔隙率、生物力學(xué)強(qiáng)度和物相容性,在基質(zhì)成分上與正常椎間盤(pán)細(xì)胞外基質(zhì)類(lèi)似,可作為組織工程椎間盤(pán)的支架材料。 3.以皮質(zhì)骨-松質(zhì)骨環(huán)制備的雙相纖維環(huán)支架具有一定的發(fā)展前景。
[Abstract]:Objective: to use natural pig bone decalcification matrix gelatin (BMG, mainly composed of type I collagen) and cartilage extracellular matrix (CECM, the main component of type II collagen /GAG) as material, a new type of fibrous ring nucleus pulposus biphasic stent was developed by bionics, and the feasibility of it as a scaffold for tissue engineering intervertebral disc was discussed.
Method:
1. fibrous ring scaffold: Taking the proximal cancellous bone of the pig femur, taking the outer diameter 10mm, the inner diameter 5mm, the hollow ring bone ring of the thick 3mm, and rinsing with high pressure water. The hollow ring decalcified bone matrix gelatin was made in order to make the hollow ring decalcified bone matrix gelatin as the intervertebral disc scaffold. Mechanical tests were carried out using a micro mechanical testing machine. The isolation and culture of rabbit adipose derived stem cells and MTT method were used to analyze the toxicity of the scaffold extract.
2. fibrous ring and nucleus pulposus biphasic stent: the porcine articular cartilage was crushed by gradient centrifugation to select 5 m ~ 100 m cartilage microfilament, and the mass volume ratio was 3.5%. The suspension was injected into the central cold freeze dry of the hollow ring decalcified bone matrix fibrous ring scaffold, and the UV and chemical crosslinking agent carbon was used. The scaffolds were crosslinked with two imines. The pore size, porosity and water absorption of the scaffold were measured by histological and scanning electron microscopy. The rabbit adipose stem cells were planted on the scaffold, inverted microscope and scanning electron microscope were used to observe the adhesion and growth of the cells on the scaffold.
3. double phase fibrous ring scaffold: Taking the central round area of the pig metacarpal bone, sawing the thick 5mm cortical bone - cancellous bone ring, rinsing, decalcification and deactivating the cortical bone - the double phase fibrous ring scaffold of the cancellous bone ring. The pore size and pore size of the scaffold were observed and the water absorption and porosity were measured. The results of mechanical test were measured by micromechanical test machine.
1. fibrous ring scaffold: the fibrous ring scaffold prepared with cancellous bone decalcified bone matrix gelatin is a milk white annular porous structure. The pore size is roughly uniform and the pores are interconnected. Under wet condition, the texture of soft.HE shows no cell structure residue in the tissue. The aperture of the scaffold is 401.44 + 13.1 with the light and scanning electron microscope. With the porosity of 62.12 + 1.52% and the water absorption rate of 409.774 + 11.34%., the biomechanical results showed that the elastic modulus of the scaffold was 47.75 + 6.32MPa. cell proliferation curve and showed that there was no significant difference in the absorbance between the different concentrations of the scaffold and the control DMEM culture (P0.05).
2. fiber ring and nucleus pulposus biphasic stent: the fibrous ring nucleus pulposus double phase scaffold constructed with cancellous bone decalcified bone matrix gelatin and cartilage extracellular matrix is yellowish white circular porous structure, pore is interconnected, and the texture is soft under wet condition. The pore size is roughly uniform, the diameter of the ring is larger than that of nucleus pulposus, and the diameter of the nucleus pulposus is 101.3 + 22.5 m. The porosity was 83.24 + 4.31%, the water absorption rate was 421.45 + 8.43%. The overall elastic modulus of the scaffold was 53.45 + 3.21MPa.HE. The cell structure was not residual in the tissue. The light and scanning electron microscope showed that the scaffold pores were uniform. The cells adhered well on the scaffold under the inverted microscope. Good long.
3. fiber ring biphasic stent: the fibrous ring biphasic stent made with cortical bone cancellous bone ring is a light yellow circular porous structure. The pore size, porosity and water absorption are smaller than that of the cancellous bone fibrous ring scaffold, the porosity is 51.41 + 2.71%, and the water absorption rate is 350.15 + 3.21%.
Conclusion:
1. the fibrous ring scaffolds prepared with cancellous bone decalcified bone matrix gelatin have good porosity, pore size, water absorption, biocompatibility, and modulus of elasticity with the fiber ring at the same order of magnitude.
2. the fibrous ring nucleus pulposus biphasic scaffold prepared with cancellous bone decalcification matrix gelatin and cartilage extracellular matrix has good pore size, porosity, biomechanical strength and substance compatibility. It is similar to the extracellular matrix of normal intervertebral disc on the matrix component. It can be used as a scaffold material for tissue engineering intervertebral disc.
3. the biphasic fiber ring scaffolds made from cortical bone cancellous bone rings have certain development prospects.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.08
本文編號(hào)：2154598
[Abstract]:Objective: to use natural pig bone decalcification matrix gelatin (BMG, mainly composed of type I collagen) and cartilage extracellular matrix (CECM, the main component of type II collagen /GAG) as material, a new type of fibrous ring nucleus pulposus biphasic stent was developed by bionics, and the feasibility of it as a scaffold for tissue engineering intervertebral disc was discussed.
Method:
1. fibrous ring scaffold: Taking the proximal cancellous bone of the pig femur, taking the outer diameter 10mm, the inner diameter 5mm, the hollow ring bone ring of the thick 3mm, and rinsing with high pressure water. The hollow ring decalcified bone matrix gelatin was made in order to make the hollow ring decalcified bone matrix gelatin as the intervertebral disc scaffold. Mechanical tests were carried out using a micro mechanical testing machine. The isolation and culture of rabbit adipose derived stem cells and MTT method were used to analyze the toxicity of the scaffold extract.
2. fibrous ring and nucleus pulposus biphasic stent: the porcine articular cartilage was crushed by gradient centrifugation to select 5 m ~ 100 m cartilage microfilament, and the mass volume ratio was 3.5%. The suspension was injected into the central cold freeze dry of the hollow ring decalcified bone matrix fibrous ring scaffold, and the UV and chemical crosslinking agent carbon was used. The scaffolds were crosslinked with two imines. The pore size, porosity and water absorption of the scaffold were measured by histological and scanning electron microscopy. The rabbit adipose stem cells were planted on the scaffold, inverted microscope and scanning electron microscope were used to observe the adhesion and growth of the cells on the scaffold.
3. double phase fibrous ring scaffold: Taking the central round area of the pig metacarpal bone, sawing the thick 5mm cortical bone - cancellous bone ring, rinsing, decalcification and deactivating the cortical bone - the double phase fibrous ring scaffold of the cancellous bone ring. The pore size and pore size of the scaffold were observed and the water absorption and porosity were measured. The results of mechanical test were measured by micromechanical test machine.
1. fibrous ring scaffold: the fibrous ring scaffold prepared with cancellous bone decalcified bone matrix gelatin is a milk white annular porous structure. The pore size is roughly uniform and the pores are interconnected. Under wet condition, the texture of soft.HE shows no cell structure residue in the tissue. The aperture of the scaffold is 401.44 + 13.1 with the light and scanning electron microscope. With the porosity of 62.12 + 1.52% and the water absorption rate of 409.774 + 11.34%., the biomechanical results showed that the elastic modulus of the scaffold was 47.75 + 6.32MPa. cell proliferation curve and showed that there was no significant difference in the absorbance between the different concentrations of the scaffold and the control DMEM culture (P0.05).
2. fiber ring and nucleus pulposus biphasic stent: the fibrous ring nucleus pulposus double phase scaffold constructed with cancellous bone decalcified bone matrix gelatin and cartilage extracellular matrix is yellowish white circular porous structure, pore is interconnected, and the texture is soft under wet condition. The pore size is roughly uniform, the diameter of the ring is larger than that of nucleus pulposus, and the diameter of the nucleus pulposus is 101.3 + 22.5 m. The porosity was 83.24 + 4.31%, the water absorption rate was 421.45 + 8.43%. The overall elastic modulus of the scaffold was 53.45 + 3.21MPa.HE. The cell structure was not residual in the tissue. The light and scanning electron microscope showed that the scaffold pores were uniform. The cells adhered well on the scaffold under the inverted microscope. Good long.
3. fiber ring biphasic stent: the fibrous ring biphasic stent made with cortical bone cancellous bone ring is a light yellow circular porous structure. The pore size, porosity and water absorption are smaller than that of the cancellous bone fibrous ring scaffold, the porosity is 51.41 + 2.71%, and the water absorption rate is 350.15 + 3.21%.
Conclusion:
1. the fibrous ring scaffolds prepared with cancellous bone decalcified bone matrix gelatin have good porosity, pore size, water absorption, biocompatibility, and modulus of elasticity with the fiber ring at the same order of magnitude.
2. the fibrous ring nucleus pulposus biphasic scaffold prepared with cancellous bone decalcification matrix gelatin and cartilage extracellular matrix has good pore size, porosity, biomechanical strength and substance compatibility. It is similar to the extracellular matrix of normal intervertebral disc on the matrix component. It can be used as a scaffold material for tissue engineering intervertebral disc.
3. the biphasic fiber ring scaffolds made from cortical bone cancellous bone rings have certain development prospects.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.08
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