足底软组织结构体外构建的初步研究
发布时间：2018-09-11 09:10
【摘要】： 足底软组织创伤在临床上比较常见,尽管创伤外科修复重建足底软组织缺损的术式比较成熟,但是对术后功能的恢复仍不满意。在足底软组织缺损修复重建中,多选用小腿部皮瓣转移修复。由于该皮瓣缺乏皮肤与深筋膜之间的纵行胶原纤维束,修复后局部软组织稳定性差,使得患者康复后站立不稳,行走打滑。恢复足底软组织特有的功能,尤其是稳定性,是临床难以解决的问题。 组织工程学是应用生命科学和工程学原理与方法,研究、开发用于修复、增进或改善人体各种组织和器官损伤后的功能和形态的一门学科。它改变了传统“以创伤修复创伤”的治疗模式,为最终实现无损伤修复创伤开辟了新途径。本研究利用组织工程学理论与技术探讨修复足底软组织创伤,以解决局部软组织稳定性问题。 足底皮下软组织结构中主要有成纤维细胞、脂肪细胞以及少量未分化的间充质细胞等。其中成纤维细胞数量较多,也是产生胶原纤维、弹性纤维和网状纤维的主要细胞。这些纤维中胶原纤维数量最多,而Ⅰ型胶原纤维又占多数。有资料显示,胶原纤维的集束还可以进行调节。因此在体外构建足底软组织过程中成纤维细胞的活性以及Ⅰ型胶原蛋白分泌量和纤维束的方向就显得至关重要。大量的研究资料表明,PTGF-BB、TGF-β1、IL-13等细胞因子对成纤维细胞具趋化作用、促进增殖和胶原蛋白的分泌,因此本实验选用成纤维细胞作为种子细胞。为使成纤维细胞高分泌Ⅰ型胶原蛋白,实验初步选用PTGF-BB、TGF-β1、IL-13三个细胞因子作为研究对象。 组织工程的另一个重要研究方向是支架材料,理想的生物支架材料应满足：模仿天然细胞外基质的结构和生物学功能；有良好的生物相容性使细胞可粘附和增殖；降解速率与组织再生的速率相匹配；适当的机械性能支撑细胞生长；具有较好的显微结构,如适合的孔隙大小、高孔隙率和孔的形态；特定的三维外形；高表面积和合适的表面理化性质。天然细胞外基质的胶原蛋白纤维尺寸为50-500 nm,因此纳米尺度支架材料能最大程度地模仿体内细胞外基质的特点。目前静电纺丝是惟一能直接、连续制备聚合物纳米纤维的方法。所以本实验采用静电纺丝法制作聚己内酯/明胶(PCL/gelatin)复合纳米纤维作为支架材料,它不仅具有表面积大、孔径小、孔隙率高、较好的均一性,直径与体内细胞外基质中胶原纤维相近,能很好的模仿细胞外基质的天然结构。而且还避免了单用PCL导致降解慢,生物相容性差,和单用明胶导致降解太快的问题。通过调节PCL、gelatin之间不同的混合比例,从而得到我们需要的降解率和生物相容性。 本实验采用出生2天的SD大鼠背部皮肤做原代培养,以获得种子细胞。应用不同的细胞因子作用于种子细胞,以研究这些细胞因子对成纤维细胞的增殖及分泌Ⅰ型胶原蛋白的影响程度。将种子细胞与PCL/gelatin纳米支架材料复合通过观察种子细胞在材料表面的生长情况和检测培养液中Ⅰ型胶原蛋白的浓度,初步的评价PCL/gelatin纳米支架材料的生物相容性和应用到本课题的可行性以及为以后在大鼠体内实验和临床应用提供实验参考。实验主要有以下两个部分： 目的：选用不同浓度的血小板源性生长因子BB (PDGF-BB)、白介素13(IL-13)、转化生长因子β1 (TGF-β1)在体外诱导真皮成纤维细胞,以研究不同的细胞因子对成纤维细胞的增殖及分泌Ⅰ型胶原蛋白的影响程度。 方法：选用出生2d的SD大鼠背部皮肤进行真皮成纤维细胞原代培养,采用免疫荧光技术对其进行纯度鉴定。实验分为四组,分别是PDGF-BB (30ng/ml)组,IL-13(100ng/m)组,TGF-β1 (10ng/ml)组和不加任何处理因素的阴性对照组,用MTT法、ELISA检测在24h、48h、72h时的真皮成纤维细胞的增殖情况及培养液中Ⅰ型胶原蛋白的浓度。用RT-PCR法检测各组中真皮成纤维细胞Ⅰ型胶原蛋白基因的表达情况。 结果：原代培养的真皮成纤维细胞的纯度达90%以上。PDGF-BB、IL-13、TGF-β1均能促进真皮成纤维细胞增殖和Ⅰ型胶原蛋白分泌,在48h,72h时PDGF-BB组的培养液中Ⅰ型胶原蛋白浓度显著高于IL-13组,TGF-β1组及阴性对照组(P0.05)。初步RT-PCR分析显示PDGF-BB组Ⅰ型胶原蛋白基因表达相对多于其它各组及对照组。 结论：PDGF-BB、IL-13、TGF-β1均能促进真皮成纤维细胞增殖和分泌Ⅰ型胶原蛋白,其中浓度为30ng/ml的PDGF-BB的作用更为显著。 目的：通过观察真皮成纤维细胞在聚己内酯/明胶(PCL/gelatin)复合纳米支架材料上生长情况以及检测培养液中的Ⅰ型胶原蛋白的浓度从而初步的评价PCL/gelatin复合纳米纤维的生物相容性。 方法：采用静电纺丝法制备PCL/gelatin复合纳米纤维,将原代培养的真皮成纤维细胞接种到材料的表面,用扫描电镜观察真皮成纤维细胞在材料表面的生长情况。实验分为3组：支架材料组、PDGF-BB组、阴性对照组,应用ELISA法检测细胞液中Ⅰ型胶原蛋白的浓度,采用重复测量方差分析和LSD多重比较法比较各组培养液中的Ⅰ型胶原蛋白浓度。 结果：PCL、gelatin混合溶液用静电纺技术制备出的PCL/gelatin纳米纤维呈现相互连通的三维网络状结构；纤维的直径与分布范围较均匀,其直径为700nm左右。真皮成纤维细胞在PCL/gelatin纳米支架材料表面贴附牢固,具有良好的生长形态,呈现长梭形或者多角形,伸展充分。细胞与纤维之间、细胞与细胞之间均有较多树枝状的突起相互交连,还可观察到细胞向纤维材料内部长入的现象。培养液中Ⅰ型胶原蛋白的检测结果统计显示,培养24h后各组间培养液中的Ⅰ型胶原蛋白的浓度无显著差异(P＞0.05)。在培养48h,72h后,PDGF-BB组培养液中的Ⅰ型胶原蛋白的浓度显著高于支架材料组和对照组(P0.05)。支架材料组与对照组在不同时间培养液中Ⅰ型胶原蛋白的浓度均无显著性差异(P0.05)。 结论：PCL/gelatin复合纳米材料具有较好的生物相容性,可以作为足底皮下软组织构建的潜在支架材料。
[Abstract]:Trauma of plantar soft tissue is common in clinic. Although the surgical method of repairing and reconstructing plantar soft tissue defect is mature, the functional recovery is still unsatisfactory. The instability of the local soft tissue after the repair of the fibrous tract makes the patient stand unstable and walk slippery after recovery. It is difficult to restore the unique function of the plantar soft tissue, especially the stability.
Tissue engineering is a subject that applies the principles and methods of life science and engineering to study and develop for repairing, enhancing or improving the function and morphology of human tissues and organs after injury. To study the application of tissue engineering theory and technology in repairing plantar soft tissue trauma and to solve the problem of local soft tissue stability.
There are mainly fibroblasts, adipocytes and a few undifferentiated mesenchymal cells in the subcutaneous tissue of plantar. Among them, fibroblasts are the main cells producing collagen fibers, elastic fibers and reticular fibers. The results indicate that the aggregation of collagen fibers can also be regulated. Therefore, the activity of fibroblasts and the secretion of collagen type I and the direction of fibrous bundles are very important in the process of plantar soft tissue construction in vitro. In order to make fibroblasts secrete type I collagen, three cytokines, PTGF-BB, TGF-beta 1 and IL-13, were selected as the research objects.
Another important research direction of tissue engineering is scaffold material, which should be satisfied with: mimic the structure and biological function of natural extracellular matrix; have good biocompatibility to enable cells to adhere and proliferate; degradation rate matches the rate of tissue regeneration; appropriate mechanical properties to support cell growth. It has good microstructure, such as suitable pore size, high porosity and pore morphology, specific three-dimensional shape, high surface area and suitable surface physical and chemical properties. At present, electrospinning is the only direct and continuous method to prepare polymer nanofibers. In this experiment, polycaprolactone/gelatin (PCL/gelatin) composite nanofibers were prepared by electrospinning as scaffolds. It has not only large surface area, small pore size, high porosity, good uniformity, diameter and in vivo and in vitro matrix gum. By adjusting the mixing ratio of PCL and gelatin, we can get the degradation rate and biocompatibility we need.
Seed cells were obtained from the back skin of two-day-old SD rats by primary culture. Seed cells were treated with different cytokines to study the effects of these cytokines on the proliferation and secretion of type I collagen in fibroblasts. The growth of seed cells on the surface of the material and the concentration of collagen type I in the culture medium were measured. The biocompatibility of PCL/gelatin nano-scaffolds and the feasibility of its application in this study were preliminarily evaluated. The experiment provided experimental reference for the future experiment and clinical application in rats.
AIM: To investigate the effects of platelet-derived growth factor BB (PDGF-BB), interleukin-13 (IL-13) and transforming growth factor-beta 1 (TGF-beta 1) on the proliferation and type I collagen secretion of fibroblasts in vitro.
METHODS: Dermal fibroblasts were cultured on the dorsal skin of SD rats born 2 days after birth and purified by immunofluorescence technique. The experiment was divided into four groups: PDGF-BB (30ng/ml), IL-13 (100ng/m), TGF-beta 1 (10ng/ml) and negative control group without any treatment factors. MTT and ELISA were used to detect the purity of dermal fibroblasts at 24h and 48h. The proliferation of dermal fibroblasts at 72 h and the concentration of collagen type I in culture medium were detected by RT-PCR.
Results: The purity of primary cultured dermal fibroblasts was over 90%. PDGF-BB, IL-13, TGF-beta 1 could promote the proliferation of dermal fibroblasts and the secretion of type I collagen. The concentration of type I collagen in culture medium of PDGF-BB group was significantly higher than that of IL-13 group at 48h and 72h, TGF-beta 1 group and negative control group (P 0.05). Preliminary RT-PCR analysis showed that P The expression of type I collagen gene in group DGF-BB was more than that in other groups and control groups.
Conclusion: PDGF-BB, IL-13 and TGF-beta 1 can promote the proliferation and secretion of type I collagen in dermal fibroblasts, and the effect of PDGF-BB at the concentration of 30 ng/ml is more significant.
AIM: To evaluate the biocompatibility of PCL/gelatin composite nanofibers by observing the growth of dermal fibroblasts on polycaprolactone/gelatin (PCL/gelatin) composite nanoscaffolds and detecting the concentration of collagen type I in culture medium.
METHODS: PCL/gelatin composite nanofibers were prepared by electrospinning method. The primary cultured dermal fibroblasts were seeded onto the surface of the material and the growth of the dermal fibroblasts on the surface of the material was observed by scanning electron microscopy. The concentration of type I collagen in culture medium was compared by repeated analysis of variance and LSD multiple comparison.
Results: PCL/gelatin nanofibers prepared by electrospinning with PCL and gelatin mixed solution showed a three-dimensional interconnected network structure; the diameter and distribution of the fibers were uniform, and the diameter of the fibers was about 700 nm. Dermal fibroblasts adhered firmly to the surface of PCL/gelatin nano-scaffolds with good growth morphology. There are many dendritic processes between cells and fibers, and there are many dendritic processes between cells and cells. The phenomena of cells growing into fibrous materials can also be observed. The concentration of type I collagen in PDGF-BB group was significantly higher than that in scaffold material group and control group at 48h and 72h after culture (P > 0.05).
CONCLUSION: PCL/gelatin composite nanomaterials have good biocompatibility and can be used as potential scaffolds for plantar subcutaneous soft tissue construction.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R322
本文编号：2236257
[Abstract]:Trauma of plantar soft tissue is common in clinic. Although the surgical method of repairing and reconstructing plantar soft tissue defect is mature, the functional recovery is still unsatisfactory. The instability of the local soft tissue after the repair of the fibrous tract makes the patient stand unstable and walk slippery after recovery. It is difficult to restore the unique function of the plantar soft tissue, especially the stability.
Tissue engineering is a subject that applies the principles and methods of life science and engineering to study and develop for repairing, enhancing or improving the function and morphology of human tissues and organs after injury. To study the application of tissue engineering theory and technology in repairing plantar soft tissue trauma and to solve the problem of local soft tissue stability.
There are mainly fibroblasts, adipocytes and a few undifferentiated mesenchymal cells in the subcutaneous tissue of plantar. Among them, fibroblasts are the main cells producing collagen fibers, elastic fibers and reticular fibers. The results indicate that the aggregation of collagen fibers can also be regulated. Therefore, the activity of fibroblasts and the secretion of collagen type I and the direction of fibrous bundles are very important in the process of plantar soft tissue construction in vitro. In order to make fibroblasts secrete type I collagen, three cytokines, PTGF-BB, TGF-beta 1 and IL-13, were selected as the research objects.
Another important research direction of tissue engineering is scaffold material, which should be satisfied with: mimic the structure and biological function of natural extracellular matrix; have good biocompatibility to enable cells to adhere and proliferate; degradation rate matches the rate of tissue regeneration; appropriate mechanical properties to support cell growth. It has good microstructure, such as suitable pore size, high porosity and pore morphology, specific three-dimensional shape, high surface area and suitable surface physical and chemical properties. At present, electrospinning is the only direct and continuous method to prepare polymer nanofibers. In this experiment, polycaprolactone/gelatin (PCL/gelatin) composite nanofibers were prepared by electrospinning as scaffolds. It has not only large surface area, small pore size, high porosity, good uniformity, diameter and in vivo and in vitro matrix gum. By adjusting the mixing ratio of PCL and gelatin, we can get the degradation rate and biocompatibility we need.
Seed cells were obtained from the back skin of two-day-old SD rats by primary culture. Seed cells were treated with different cytokines to study the effects of these cytokines on the proliferation and secretion of type I collagen in fibroblasts. The growth of seed cells on the surface of the material and the concentration of collagen type I in the culture medium were measured. The biocompatibility of PCL/gelatin nano-scaffolds and the feasibility of its application in this study were preliminarily evaluated. The experiment provided experimental reference for the future experiment and clinical application in rats.
AIM: To investigate the effects of platelet-derived growth factor BB (PDGF-BB), interleukin-13 (IL-13) and transforming growth factor-beta 1 (TGF-beta 1) on the proliferation and type I collagen secretion of fibroblasts in vitro.
METHODS: Dermal fibroblasts were cultured on the dorsal skin of SD rats born 2 days after birth and purified by immunofluorescence technique. The experiment was divided into four groups: PDGF-BB (30ng/ml), IL-13 (100ng/m), TGF-beta 1 (10ng/ml) and negative control group without any treatment factors. MTT and ELISA were used to detect the purity of dermal fibroblasts at 24h and 48h. The proliferation of dermal fibroblasts at 72 h and the concentration of collagen type I in culture medium were detected by RT-PCR.
Results: The purity of primary cultured dermal fibroblasts was over 90%. PDGF-BB, IL-13, TGF-beta 1 could promote the proliferation of dermal fibroblasts and the secretion of type I collagen. The concentration of type I collagen in culture medium of PDGF-BB group was significantly higher than that of IL-13 group at 48h and 72h, TGF-beta 1 group and negative control group (P 0.05). Preliminary RT-PCR analysis showed that P The expression of type I collagen gene in group DGF-BB was more than that in other groups and control groups.
Conclusion: PDGF-BB, IL-13 and TGF-beta 1 can promote the proliferation and secretion of type I collagen in dermal fibroblasts, and the effect of PDGF-BB at the concentration of 30 ng/ml is more significant.
AIM: To evaluate the biocompatibility of PCL/gelatin composite nanofibers by observing the growth of dermal fibroblasts on polycaprolactone/gelatin (PCL/gelatin) composite nanoscaffolds and detecting the concentration of collagen type I in culture medium.
METHODS: PCL/gelatin composite nanofibers were prepared by electrospinning method. The primary cultured dermal fibroblasts were seeded onto the surface of the material and the growth of the dermal fibroblasts on the surface of the material was observed by scanning electron microscopy. The concentration of type I collagen in culture medium was compared by repeated analysis of variance and LSD multiple comparison.
Results: PCL/gelatin nanofibers prepared by electrospinning with PCL and gelatin mixed solution showed a three-dimensional interconnected network structure; the diameter and distribution of the fibers were uniform, and the diameter of the fibers was about 700 nm. Dermal fibroblasts adhered firmly to the surface of PCL/gelatin nano-scaffolds with good growth morphology. There are many dendritic processes between cells and fibers, and there are many dendritic processes between cells and cells. The phenomena of cells growing into fibrous materials can also be observed. The concentration of type I collagen in PDGF-BB group was significantly higher than that in scaffold material group and control group at 48h and 72h after culture (P > 0.05).
CONCLUSION: PCL/gelatin composite nanomaterials have good biocompatibility and can be used as potential scaffolds for plantar subcutaneous soft tissue construction.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R322
【参考文献】
相关期刊论文 前10条
1 任雨笙,黄佐,潘晓明,杜荣增,顾兴建,吴宗贵;血小板衍生生长因子-BB对血管内皮细胞、平滑肌细胞及成纤维细胞胶原蛋白合成的影响[J];第二军医大学学报;2005年02期
2 鲍扬波;王家俊;胡巧玲;;聚合物静电纺及在组织工程支架中的应用[J];纺织学报;2008年02期
3 刘晓军;苏利国;刘勇;段家波;张晓军;樊利军;张会勇;;足底内侧动脉岛状皮瓣治疗足跟部皮肤缺损[J];中国骨与关节损伤杂志;2007年08期
4 吴林波,丁建东;组织工程三维多孔支架的制备方法和技术进展[J];功能高分子学报;2003年01期
5 张爱英,王连才,刘厚利,魏宏亮,冯增国;基于快速成型技术的组织工程支架制备进展[J];化工进展;2004年03期
6 胡永斌;曾庆富;冯德云;李翔;彭劲武;;激活蛋白-1调控转化生长因子β_1诱导的人肺成纤维细胞Ⅰ型胶原分泌[J];中南大学学报(医学版);2007年05期
7 张丽娟;李倩;陈萍;闫静波;王瑞敏;杨嫣;杨方;;细胞外信号调节激酶1/2途径在血小板衍生生长因子诱导的大鼠心脏成纤维细胞增殖与胶原合成中的调节作用[J];解剖学杂志;2007年04期
8 肖刚;王智园;谭敏;胡少为;石灵春;;转化生长因子β_1对病理性瘢痕中成纤维细胞增殖及凋亡水平的影响[J];实用医学杂志;2008年13期
9 胡葵葵;戴育成;袁敬东;;组织工程活性真皮的构建研究[J];西安交通大学学报(医学版);2006年02期
10 熊丽霞;石小玉;李文林;周莹;唐洪林;;白细胞介素13刺激成纤维细胞胶原基因转录和胶原蛋白的合成[J];中国组织工程研究与临床康复;2007年23期
，本文编号：2236257
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2236257.html
最近更新
教材专著
