组织工程脱细胞气管支架细胞复合物的生物安全性评价
发布时间：2018-09-09 09:56
【摘要】：目的： 一、利用京尼平交联脱氧胆酸钠酶联合法与Trixon-100酶联合法,分别结合BMMSCs制备组织工程脱细胞气管支架细胞复合物。 二、对组织工程脱细胞气管支架细胞复合物的急性毒性、细胞毒性、血管化能力、体内免疫炎性反应及支架降解被吸收情况检测评价。 三、通过对组织工程脱细胞气管支架细胞复合物的生物安全性能进行评价,为制备更接近原生气管的替代物提供实验依据。 方法： 一、组织工程脱细胞气管支架细胞复合物的制备： ①细胞部分：将2月龄大体重约1.2kg的健康新西兰大白兔一只作为BMMSCs供体,经原代及传代培养获得第3代BMMSCs。 ②脱细胞气管支架的制备：将5只4月龄体重在2.5kg健康新西兰大白兔作为气管支架的供体,分别用京尼平交联脱氧胆酸钠酶联合法与Trixon-100酶联合法对取材的气管进行脱细胞处理获得脱细胞气管支架。 ③脱细胞气管支架细胞复合物的制备：将BMMSCs分别种植于上述两种气管支架外表面并培养。 ④实验分组：京尼平交联脱氧胆酸钠酶联合法结合BMMSCs (5x104cells/ml)制备的组织工程脱细胞气管支架细胞复合物作为实验A组,Trixon-100酶联合法结合BMMSCs制备组织工程脱细胞气管支架细胞复合物后者作为实验B组。 二、通过以下5种方法检测组织工程脱细胞气管支架细胞复合物的生物安全性： ①将A组浸渍液、B组浸渍液及阴性组(生理盐水)注入小鼠腹腔检测两种脱细胞气管支架细胞复合物对小鼠毒性； ②应用MTT法检测A组、B组、阴性对照组(聚全氟乙丙烯)及阳性对照组(聚氟乙烯)中BMMSCs生长增殖情况； ③通过SEM(扫描电子显微镜检查)来观察两实验组表面的BMMSCs粘附生长状态; ④对A组、B组、阴性组(戊二醛浸润的A组)及阳性组(明胶海绵)应用鸡胚绒毛膜实验观察脱细胞气管支架细胞复合物的血管化程度； ⑤以原BMMSCs供体大白兔作为移植受体,将这两实验组分别于移植于受体皮下脊柱两侧。移植后第7、14、28d取出脱细胞气管支架细胞复合物,与移植前的脱细胞气管支架细胞复合物,分别进行连续形态学观察及HE染色,以检测免疫炎症反应。 结果： 一、脱细胞气管支架细胞复合物浸渍液注入小鼠腹腔后,三组小鼠活动度、进食及排便均正常,体重增加(p0.05)及呼吸频率变化(p0.05)无统计学意义。 二、细胞形态学观察发现两实验组上的BMMSCs均贴壁生长良好,无明显差异。 三、MTT检测绘制细胞生长曲线,发现阴性对照组细胞生长最好,阴性组最差,B组细胞增长比A组稍快,但无明显区别。 四、扫描电子显微镜：两组BMMSCs均完全粘附覆盖支架外表面,细胞层叠,连接紧密。 五、鸡胚绒毛膜实验观察显示：A组、B组及阳性组周围均见新生血管形成,以阳性组最明显,A组新生血管较多,部分新生血管爬行于脱细胞气管支架复合物底面,B组新生血管少于A组,阴性组周围未见明显血管形成。 六、通过四次(移植前、移植后第7、14、28d脱细胞气管支架细胞复合物)连续组织学检测及HE染色发现：A组细胞支架复合物仍为暗红色,硬度无减弱,无明显炎症,细胞支架复合物各层结构完整,无降解；B组细胞支架复合物呈淡红色,硬度减弱,炎症较重,支架各层结构混乱,逐渐软化降解。 结论： 一、A组与B组均无急性毒性及细胞毒性反应,均可促进细胞生长增殖及支架基质血管再生,且A组血管化能力更强。 二、A组不引起炎症反应、不易软化降解的性能更有利于移植体在体内长期存活及功能作用。
[Abstract]:Objective:
Firstly, the acellular tracheal scaffold cell complex was prepared by Genipin cross-linked deoxycholate sodium enzyme combined with Trixon-100 enzyme combined with BMMSCs.
Secondly, the acute toxicity, cytotoxicity, vascularization ability, in vivo immune inflammatory reaction and scaffold degradation and absorption of acellular tracheal scaffold complex were detected and evaluated.
Thirdly, the biosafety of acellular tracheal scaffold cell complex was evaluated to provide experimental basis for the preparation of substitutes closer to the original gas tube.
Method:
(1) preparation of tissue engineered acellular tracheal scaffold complex:
(1) Cell part: A 2-month-old healthy New Zealand white rabbit weighing about 1.2 kg was used as a BMMSCs donor. The third generation of BMMSCs were obtained by primary culture and subculture.
(2) Preparation of acellular tracheal stents: Five 4-month-old healthy New Zealand white rabbits weighing 2.5 kg were used as tracheal stent donors. The acellular tracheal stents were obtained by acellular treatment with Genipin crosslinked deoxycholate enzyme and Trixon-100 enzyme respectively.
(3) Preparation of acellular tracheal scaffold cell complex: BMMSCs were implanted on the surface of the two kinds of tracheal scaffolds and cultured.
(4) Experimental grouping: The tissue engineered acellular tracheal scaffold cell complex prepared by Genipin cross-linked deoxycholate enzyme combined with BMMSCs (5x104 cells/ml) was used as experimental group A, and the tissue engineered acellular tracheal scaffold cell complex prepared by Trixon-100 enzyme combined with BMMSCs as experimental group B.
Secondly, the biosafety of tissue engineered acellular tracheal scaffold cell complex was detected by the following five methods:
The toxicity of two acellular tracheal scaffold cell complexes was detected by injecting group A, group B and negative group (normal saline) into the abdominal cavity of mice.
(2) The growth and proliferation of BMMSCs in group A, group B, negative control group (PFE) and positive control group (PFE) were detected by MTT.
(3) observe the adherence growth state of BMMSCs on the two experimental group by SEM (scanning electron microscopy).
(4) The vascularization of acellular tracheal scaffold cell complex was observed in group A, group B, negative group (glutaraldehyde infiltration group A) and positive group (gelatin sponge).
_The two experimental groups were transplanted into the subcutaneous spine of the recipient with the original BMMSCs donor rabbit as the recipient. On the 7th, 14th and 28th day after transplantation, the acellular tracheal scaffold cell complex was taken out and the acellular tracheal scaffold cell complex was observed and stained with HE to detect the immunoinflammatory reaction.
Result:
Firstly, after the acellular tracheal scaffold cell complex impregnated solution was injected into the abdominal cavity of mice, the activity, food intake and defecation of the three groups of mice were normal, weight gain (p0.05) and respiratory rate changes (p0.05) were not statistically significant.
Two, cell morphology observation showed that two of BMMSCs in experimental group adhered well and had no significant difference.
Third, MTT assay plotted the cell growth curve, found that the negative control group had the best cell growth, the negative group had the worst cell growth, the B group cell growth was slightly faster than the A group, but there was no significant difference.
Fourthly, scanning electron microscopy: BMMSCs in both groups completely adhered to the outer surface of the scaffold, and the cells overlapped tightly.
Fifth, the chicken embryo chorionic membrane experiment showed that neovascularization was observed in group A, group B and around the positive group. The most obvious neovascularization was found in the positive group. There were more neovascularization in group A. Some of the neovascularization crawled on the bottom of the acellular tracheal stent complex.
Sixthly, four times (before transplantation, 7, 14, 28 days after transplantation) continuous histological examination and HE staining showed that the scaffold complex in group A was still dark red, the stiffness was not weakened, there was no obvious inflammation, the structure of the scaffold complex was intact, and there was no degradation; the scaffold complex in group B was light red, and the stiffness was reduced. The structure of the scaffold is chaotic and gradually softened and degraded.
Conclusion:
First, both group A and group B had no acute toxicity and cytotoxicity. Both groups could promote cell growth and proliferation and angiogenesis of scaffold matrix, and group A had stronger vascularization ability.
Secondly, group A did not cause inflammation and was not easy to soften and degrade, which was more conducive to the long-term survival and function of the graft in vivo.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08
本文编号：2232050
[Abstract]:Objective:
Firstly, the acellular tracheal scaffold cell complex was prepared by Genipin cross-linked deoxycholate sodium enzyme combined with Trixon-100 enzyme combined with BMMSCs.
Secondly, the acute toxicity, cytotoxicity, vascularization ability, in vivo immune inflammatory reaction and scaffold degradation and absorption of acellular tracheal scaffold complex were detected and evaluated.
Thirdly, the biosafety of acellular tracheal scaffold cell complex was evaluated to provide experimental basis for the preparation of substitutes closer to the original gas tube.
Method:
(1) preparation of tissue engineered acellular tracheal scaffold complex:
(1) Cell part: A 2-month-old healthy New Zealand white rabbit weighing about 1.2 kg was used as a BMMSCs donor. The third generation of BMMSCs were obtained by primary culture and subculture.
(2) Preparation of acellular tracheal stents: Five 4-month-old healthy New Zealand white rabbits weighing 2.5 kg were used as tracheal stent donors. The acellular tracheal stents were obtained by acellular treatment with Genipin crosslinked deoxycholate enzyme and Trixon-100 enzyme respectively.
(3) Preparation of acellular tracheal scaffold cell complex: BMMSCs were implanted on the surface of the two kinds of tracheal scaffolds and cultured.
(4) Experimental grouping: The tissue engineered acellular tracheal scaffold cell complex prepared by Genipin cross-linked deoxycholate enzyme combined with BMMSCs (5x104 cells/ml) was used as experimental group A, and the tissue engineered acellular tracheal scaffold cell complex prepared by Trixon-100 enzyme combined with BMMSCs as experimental group B.
Secondly, the biosafety of tissue engineered acellular tracheal scaffold cell complex was detected by the following five methods:
The toxicity of two acellular tracheal scaffold cell complexes was detected by injecting group A, group B and negative group (normal saline) into the abdominal cavity of mice.
(2) The growth and proliferation of BMMSCs in group A, group B, negative control group (PFE) and positive control group (PFE) were detected by MTT.
(3) observe the adherence growth state of BMMSCs on the two experimental group by SEM (scanning electron microscopy).
(4) The vascularization of acellular tracheal scaffold cell complex was observed in group A, group B, negative group (glutaraldehyde infiltration group A) and positive group (gelatin sponge).
_The two experimental groups were transplanted into the subcutaneous spine of the recipient with the original BMMSCs donor rabbit as the recipient. On the 7th, 14th and 28th day after transplantation, the acellular tracheal scaffold cell complex was taken out and the acellular tracheal scaffold cell complex was observed and stained with HE to detect the immunoinflammatory reaction.
Result:
Firstly, after the acellular tracheal scaffold cell complex impregnated solution was injected into the abdominal cavity of mice, the activity, food intake and defecation of the three groups of mice were normal, weight gain (p0.05) and respiratory rate changes (p0.05) were not statistically significant.
Two, cell morphology observation showed that two of BMMSCs in experimental group adhered well and had no significant difference.
Third, MTT assay plotted the cell growth curve, found that the negative control group had the best cell growth, the negative group had the worst cell growth, the B group cell growth was slightly faster than the A group, but there was no significant difference.
Fourthly, scanning electron microscopy: BMMSCs in both groups completely adhered to the outer surface of the scaffold, and the cells overlapped tightly.
Fifth, the chicken embryo chorionic membrane experiment showed that neovascularization was observed in group A, group B and around the positive group. The most obvious neovascularization was found in the positive group. There were more neovascularization in group A. Some of the neovascularization crawled on the bottom of the acellular tracheal stent complex.
Sixthly, four times (before transplantation, 7, 14, 28 days after transplantation) continuous histological examination and HE staining showed that the scaffold complex in group A was still dark red, the stiffness was not weakened, there was no obvious inflammation, the structure of the scaffold complex was intact, and there was no degradation; the scaffold complex in group B was light red, and the stiffness was reduced. The structure of the scaffold is chaotic and gradually softened and degraded.
Conclusion:
First, both group A and group B had no acute toxicity and cytotoxicity. Both groups could promote cell growth and proliferation and angiogenesis of scaffold matrix, and group A had stronger vascularization ability.
Secondly, group A did not cause inflammation and was not easy to soften and degrade, which was more conducive to the long-term survival and function of the graft in vivo.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08
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