改性组织工程材料重建尿道的实验研究

发布时间：2018-03-11 15:10

本文选题：明胶　切入点：细菌纤维素　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分明胶-细菌纤维素复合舌黏膜上皮细胞重建兔尿道的实验研究目的:评估明胶-细菌纤维素复合舌黏膜上皮细胞重建兔尿道缺损的效果。材料及方法:制备明胶-细菌纤维素生物材料,通过扫描电镜评估材料结构特征。分离兔舌黏膜细胞,细胞培养扩增至足够数量后种植在明胶-细菌纤维素支架上。建立兔尿道缺损模型(2.0×0.8cm),单纯明胶-细菌纤维素支架(对照组,N=10)和复合细胞的明胶-细菌纤维素支架(实验组,N=10)分别修复尿道缺损。在手术后的1和3个月,通过大体观察、尿道造影及组织学分析评估修复尿道缺损的效果。结果:明胶-细菌纤维素由致密状的上表面和孔隙状的下表面的双层结构构成。兔舌黏膜上皮细胞能被成功分离、培养和扩增,在支架材料上生长良好。在术后1个月,两组修复段尿道均保持通畅。但在术后3个月,对照组尿道出现狭窄,而实验组尿道仍保持通畅。通过组织学分析发现,在术后1个月,实验组修复段尿道表面有一层完整的上皮,而对照组尿道表面的上皮层不完整。但是,在术后3个月,两组尿道表面均形成多层完整的上皮,两者无统计学差异(p0.05)。在术后1和3个月,实验组尿道平滑肌和血管的再生明显优于对照组(p0.05)。另外,我们发现对照组尿道出现明显的炎症反应和纤维化。结论:我们的研究表明复合舌黏膜上皮细胞的明胶-细菌纤维素能成功修复兔尿道缺损,促进平滑肌和血管的再生,未发现明显的炎症反应和纤维化表现。复合舌黏膜上皮细胞的明胶-细菌纤维素支架有望成为一种理想材料用于尿道修复重建。第二部分三维多孔膀胱脱细胞基质在兔尿道修复重建中的应用目的:评估三维多孔膀胱脱细胞基质(BAM)修复兔尿道缺损的疗效材料及方法:在30只兔中建立腹侧尿道黏膜缺损(1.5×0.8 cm)模型。应用5%过氧乙酸(PAA)处理过的BAM(三维多孔BAM,实验组,15只)和未处理过的BAM(对照组,15只)采用补片方式分别修复兔缺损尿道。手术后1,2和3个月兔行尿道造影和组织学分析。结果:在实验组中,13只兔的尿道通畅,无尿瘘和狭窄;而在对照组中,10只兔的尿道通畅。组织学检测显示,在术后1,2和3个月,实验组尿道上皮再生的速度快于对照组;实验组平滑肌与胶原比值和平滑肌含量高于对照组;同时,血管再生优于对照组。两者比较均有统计学差异(p0.05)。结论:与未处理过的BAM相比,PAA处理过的BAM更能促进尿道重建中上皮,平滑肌和血管的再生。三维多孔BAM可能作为一种优化支架用于细胞种植的管状或长段尿道缺损的修复重建。第三部分明胶-细菌纤维素与三维多孔膀胱脱细胞基质植入兔膀胱后的组织反应研究目的:评估明胶-细菌纤维素(BC)与三维多孔膀胱脱细胞基质(BAM)植入兔膀胱后的组织反应。材料与方法:制备明胶-BC与三维多孔BAM支架材料,并评估它们的孔径和孔隙率。将20只兔膀胱后壁的对应两侧的黏膜与平滑肌剔除(1.5×2.0 cm)。然后,明胶-BC与三维多孔BAM支架材料分别植入膀胱后壁缺损处。手术后4、8周,动物被处死(每个时间检测点为10只),将植入处的膀胱组织进行大体观察,并行组织学检测与肌肉收缩分析评估两种材料植入处组织再生情况。结果:明胶-BA与三维多孔BAM支架材料的孔径与孔隙率相似,两者比较均无统计学差异(P0.05)。在术后4周,4只兔膀胱内可见膀胱结石形成,但术后8周,膀胱内未见有膀胱结石。组织分析显示,在术后4、8周,明胶-BC支架材料植入处的尿路上皮、平滑肌及血管的再生情况与三维多孔BAM相比较,均无统计学差异(P0.05)。同样,在材料植入处膀胱组织对卡巴胆碱、KCl及苯肾上腺素的收缩反应方面,两组比较均无统计学差异(P0.05)。另外,术后4、8周,两种材料植入处均未见明显的急、慢性炎症反应。结论:我们的结果提示明胶-BC与三维多孔BAM在促进膀胱组织结构和功能再生方面,具有相似的作用,均表现良好的生物相容性。明胶-BC与三维多孔BAM作为两种优化生物材料在尿道修复重建中可能具有相似的作用。
[Abstract]:The first part of the experiment study on gelatin - bacterial cellulose composite tongue epithelial cells of rabbit urethra reconstruction Objective: To evaluate the gelatin bacterial cellulose composite tongue epithelial cells to reconstruct rabbit urethral defect effect. Materials and methods: preparation of gelatin bacterial cellulose biological materials by scanning electron microscopy evaluation of material structure. Isolated rabbit tongue mucosa cells, cells after a sufficient number of cultured to grow in gelatin bacterial cellulose bracket. A rabbit model of urethral defect (2 * 0.8cm), pure bacterial cellulose gelatin scaffold (control group, N=10) and composite cell gelatin bacterial cellulose scaffolds (experimental group, N=10) respectively. The defects were repaired in 1 after surgery and 3 months by gross observation, histological analysis evaluation of urethral defect repair effect of urethrography and tissue. Results: gelatin bacterial cellulose by dense on the surface and pore shape under the table A surface of the bilayer. Rabbit lingual epithelial cells can be successfully isolated, cultured and expanded, the scaffold grew well. In 1 months after operation, two groups of repair urethra were unobstructed. But 3 months after operation, the control group and the experimental group appear urethral stricture, urethral smooth. The histological analysis showed that in the 1 months after surgery, the experimental group to repair urethra surface has a layer of intact epithelium, while the control group of the urethral epithelial layer surface is not complete. However, 3 months after operation, two groups were formed multilayer complete urethral surface epithelium, both no statistical differences (P0.05). After 1 and 3 months, the experimental group of urethral smooth muscle and vascular regeneration was significantly better than the control group (P0.05). In addition, we found that the control group appears urethral inflammation and fibrosis significantly. Conclusion: our study showed that the composite tongue epithelial cells on gelatin bacterial cellulose Can successfully repair the rabbit urethral defect, promote regeneration of smooth muscle and vascular inflammation and fibrosis, no obvious findings. Composite tongue epithelial cells on gelatin scaffold of bacterial cellulose is a kind of ideal material for urethral reconstruction. The second part application of three-dimensional porous bladder acellular matrix in rabbit urethral reconstruction. Evaluation of three-dimensional porous bladder acellular matrix (BAM) clinical materials and methods for repairing rabbit urethral defect: the establishment of ventral urethral mucosal defect in 30 rabbits (1.5 x 0.8 cm) model. Application of 5% peracetic acid (PAA) treated BAM (3D porous BAM, experimental group, 15 rats) and untreated the BAM (control group, 15 rats) by patch were used to repair defects in rabbits urethra. Analysis of 1,2 and 3 rabbit urethra angiography and tissue after surgery. Results: in the experimental group, 13 rabbits of urethral patency, no fistula and narrow Narrow; in the control group, 10 rabbits of urethral patency. Histological examination revealed that at 1,2 and 3 months postoperatively, the experimental group of urethral epithelial regeneration was faster than the control group; the experimental group and the ratio of collagen and smooth muscle smooth muscle was higher than that in control group; at the same time, angiogenesis is better than that of the control group. The two there were statistically significant differences (P0.05). Conclusion: compared with untreated BAM, PAA treated BAM can promote epithelial urethral reconstruction, and vascular smooth muscle regeneration. Three dimensional porous BAM may serve as a scaffold for the repair and reconstruction of optimal cell seeding or long tubular urethral defect. The third part of gelatin bacteria cellulose and three-dimensional porous bladder to study tissue reaction after implantation of rabbit bladder stromal cells: evaluation of gelatin bacterial cellulose (BC) and three-dimensional porous bladder acellular matrix (BAM) implanted in rabbit bladder after tissue reaction. Materials and methods Preparation of gelatin -BC and porous BAM scaffolds, and evaluate their pore size and porosity. The corresponding 20 rabbits on both sides of the posterior wall of the bladder mucosa and smooth muscle removed (1.5 x 2 cm). Then, the gelatin -BC and 3-D porous BAM scaffolds were implanted into the posterior wall of bladder defect 4,8 weeks after the operation. That animal was killed (each time the detecting point is 10), gross observation will be implanted at the bladder tissue, histological detection and muscle contraction analysis evaluation of two kinds of materials were implanted into the tissue regeneration. Results: the pore size and porosity of gelatin -BA and three-dimensional porous BAM scaffold is similar to that of the two were no statistically significant difference (P0.05). After 4 weeks, 4 rabbits were found in the bladder bladder stone formation, but 8 weeks after operation, there was no bladder bladder stones. Structure analysis showed that in 4,8 weeks after operation, -BC gelatin scaffold was implanted into the urinary tract epithelial and smooth muscle. The regeneration of blood vessels and three-dimensional porous BAM, there was no significant difference (P0.05). Similarly, in the material implanted bladder tissue of Kaba choline, KCl and contractile responses to phenylephrine, the two groups were not statistically significant (P0.05). In addition, 4,8 weeks after operation, two materials were implanted there was no obvious acute and chronic inflammation. Conclusion: Our results suggest that the gelatin -BC and three-dimensional porous structure and function of BAM in promoting the regeneration of bladder tissue, had similar effects, showed good biocompatibility. Gelatin -BC and three-dimensional porous BAM as two kinds of optimization of biological materials may have similar effects on urethral repair reconstruction.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R699

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