组织工程肌腱支架的设计及性能研究
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摘要
肌腱损伤是存在于从事体育运动人群的常见损伤之一。目前的治疗方法存在着一定的局限性或不足。组织工程技术的诞生和发展为临床肌腱修复提供了一种更为理想的、符合生理特点的方法。用组织工程技术修复肌腱缺损就是将获取少量的肌腱种子细胞在体外培养扩增后和可生物降解支架结合成复合物,将其植入缺损部位后种子细胞增殖、分化、分泌基质,形成修复组织,生物材料逐渐降解,最终达到生物学意义上的完全修复。其中支架在组织工程技术中占有非常重要的地位,它不仅起支撑作用,保持原有组织的形状,而且还起到模板作用,为细胞提供寄宿、成长、分化和增殖的场所,对受损组织的再生进行引导和对再生组织的结构进行控制,是决定组织工程技术是否能用于临床的关键因素。目前已有的组织工程肌腱支架通常有着良好的生物相容性,细胞能在支架上很好的黏附、增殖并分泌细胞外基质,但这些支架却无法满足在降解过程中的力学要求。本文设计并制备了一种新型组织工程肌腱支架,使其在满足种子细胞的黏附、增殖以及分泌细胞外基质的同时,还能够满足降解过程中的力学性能要求。
本文根据组织工程肌腱支架的设计原则,选用PGA纤维和PLA纤维作为支架材料,设计了一种将两种纤维联合使用、具有“芯-鞘”结构的新型组织工程肌腱支架,“芯”为细胞的黏附和增殖提供场所,而“鞘”是支架增强体,在组织形成前提供足够的力学强度。其中“芯”为PGA纤维须条,“鞘”为小口径管状纬平针织物。制备支架增强体的纱线采用由PGA纤维和PLA纤维组成的编织线。
本文对组织工程肌腱支架材料使用的PGA纤维和PLA纤维的基本性能、体外降解性能、细胞黏附性能进行了较为系统、全面的研究,并探讨了熔融纺丝工艺参数对PGA纤维降解性能的影响。本文研究表明,PGA纤维与PLA纤维具有良好的力学性能,可以在纺织技术中应用。PGA纤维的降解速度快于PLA纤维,在降解8周后失去几乎全部质量,在降解2周后失去绝大部分强力,在整个降解过程中表面形态发生了明显的变化,结晶度的上升说明了纤维的非结晶区降解速度较快。而PLA纤维在整个降解过程中的变化不明显。细胞在PGA纤维上的黏附要好于在PLA纤维上的黏附,并分泌了大量的细胞外基质。纺丝过程中牵伸倍数、牵伸温度和纺丝所用聚合物的特性粘度因素对PGA纤维的降解性能有着重要的影响。在实验范围内,较高牵伸倍数条件下制得的PGA纤维在降解过程中的降解速率较慢；较高牵伸温度条件下得到的PGA纤维降解速率较快；特性粘度较高的聚合物在相同纺丝条件下制得的纤维比由较低特性粘度的聚合物制得的纤维降解速率慢。
本文研究了组织工程肌腱支架增强体用编织线的制备过程,探讨了编织加工对PGA纤维降解性能的影响,还研究了编织线中不同比例的PGA纤维和PLA纤维组分对编织线降解性能的影响。研究表明,PGA纤维和PGA编织线的降解大体趋势相同,两者的降解过程均可分为“强力下降期”、“非结晶区质量损失期”、“结晶区质量损失期”三个阶段。在第一阶段,编织线中的纤维断裂强力保持率始终高于未经编织的纤维。在第二阶段,未经编织的纤维的质量损失率始终高于编织线,结晶度的增大速率也快于编织线。在第三阶段,未经编织的纤维和编织线的结晶度趋于一致,但编织线的质量损失速率较大。对4种PGA与PLA纤维成分比例不同的编织线进行体外降解试验表明,降解过程中随着编织线中PGA纤维成分比例的增大,编织线的降解速度也不断加快。
本文分别在圆机和横机上制备了作为组织工程肌腱增强体的小口径管状纬平针织物,并探讨了针织工艺参数对其几何形状、力学性能的影响。在用圆机制备组织工程肌腱增强体的过程中,弯纱深度、牵拉力和给纱张力等工艺参数对织物的几何形态以及力学性能产生影响。在弯纱深度较小、牵拉力较小以及给纱张力较大的条件下制得的织物线圈长度较小,线圈比较规整,歪斜少,结构较为紧密,断裂强力较高；而在弯纱深度较大、牵拉力较大以及给纱张力较小的条件下制得的织物线圈长度较大,结构较为稀松,断裂强力较低。在用横机制备组织工程肌腱增强体的过程中,弯纱深度、牵拉力和给纱张力等工艺参数对织物的几何形态以及力学性能的影响与在小口径圆机上一致。可通过改变横机编织针数的方法控制支架增强体直径,适用于有明确几何形态要求的组织工程肌腱支架增强体的制备。
本文对组织工程肌腱支架增强体的体外降解性能和细胞黏附性能进行了研究,建立了小口径管状纬平针织物的几何模型。研究了不同PGA和PLA纤维成分比例对组织工程肌腱支架增强体的体外降解性能和细胞黏附性能的影响,发现在支架增强体中PGA纤维成分越多,降解速率越快,在降解过程中力学性能下降程度越大,细胞在支架增强体上的黏附情况越好,优选4PGA/2PLA编织线制备支架增强体。本文建立了小口径管状纬平针织物的几何模型,对线圈的几何形态进行了分析,给出了线圈横向和纵向变形达到极限时的几何特点,并推导了表面孔隙率的计算公式。
本文制备了完整的组织工程肌腱支架,并对完整支架的性能进行了测试和研究。研究表明该“芯-鞘”结构的组织工程肌腱支架具有合适的几何形状,较高的孔隙率以及良好的力学性能。“芯-鞘”结构的组织工程肌腱支架的整个降解过程分为三个阶段,第一阶段的主要特点为强力的大幅下降,可称之为“强力下降期”;第二阶段的主要特点为质量损失率大幅上升,可称之为“质量损失期”；第三阶段,组织工程肌腱支架的质量损失率和强力保持率均较为稳定,可称之为“准稳定期”。
本文在组织工程肌腱支架上接种细胞后进行体外构建,对该支架应用于临床的可能性进行评价。构建一周后的细胞-支架复合体的断裂强力为63N；细胞-支架复合物的表面较为光滑,表层的支架增强体与中心部分的PGA纤维须条结合紧密,细胞-支架复合物的几何形状在构建过程中发生了明显的变化,其直径变细,而长度变长；细胞在支架增强体和PGA纤维须条上均黏附较好,而且分泌了大量的细胞外基质。利用该种具有“芯-鞘”结构的组织工程肌腱支架构建出的组织结构致密,胶原沿力学轴向排列,细胞／胶原比与正常肌腱近似,具有临床应用的可能性。
Injures to tendons are very common in the physically active population. However, some limitations exist in the present therapies. The tissue engineering keeps the promise for tendon repair. Using the tissue engineering for tendon repair is as follows: combined the expanded cells and a biodegradable scaffold to form a composite, and cells proliferate, differentiate and produce the extracellular matrices to form a new tissue after grafting it in the injury position. The scaffold plays a key role in tissue engineering. It not only plays a support role to keep the original tissue shape, but also plays a template role to provide a place for cell attachment, growth, proliferation and differentiation. The scaffold which can conduct the tissue regeneration and control the structure of the tissue is a key factor to determine whether the tissue engineering can be used in clinical treatment or not. The present scaffold for tendon tissue engineering possessed the good biocompatibility, and cells could attach, proliferate and produce extracellular matrices. However, those scaffolds could not meet the mechanical requirements during degradation. This paper designed and prepared a novel scaffold for tendon tissue engineering. It not only could provide a place for cell attachment, proliferation and producing extracellular matrices, but also could meet the mechanical requirements during degradation.
This paper designed a novel scaffold for the tendon tissue engineering according to the design principles of tissue engineering scaffolds. The scaffold composed of polyglycolide (PGA) and polylactide (PLA) fibers possessed a "core-sheath" structure. The "core" provided a place for cell attachment and proliferation, and the "sheath" was a scaffold reinforcement, which provided sufficient strength before tissue formation. The "core" was composed of PGA fibers, and the "sheath" was a small-diameter circular plain knitted fabric which is made from PGA/PLA braided yarns.
This paper studied the basic properties, performance during degradation in vitro, and cell-fiber reaction of PGA and PLA fiber. The results showed that both PGA and PLA fiber possessed the good mechanical properties, and could be used in textile technologies. The PGA fiber degraded faster than PLA fiber. It lost almost all the mass after 8-week degradation, but lost almost all the tensile strength after 2-week degradation. The morphology changed obviously during degradation. The crystallinity increased firstly and then decreased with the degradation time, which indicated the degradation occurred in the amorphous region. However, the PLA fiber changed not obviously during degradation. Cells attached on PGA fibers much better than on PLA fibers, and cells on PGA fibers produced more matrix. Drawing multiple, drawing temperature and inherent viscosity of polymer had the influence on the performance of PGA fiber during degradation. The PGA fiber produced on higher drawing multiple degraded more slowly. The PGA fiber produced on higher drawing temperature degraded faster. The PGA fiber made from higher inherent viscosity polymer degraded more slowly.
This paper studied the preparation of the braided yarns used in the reinforcement of scaffold for tendon tissue engineering, discussed the influence of braiding process on the performance of PGA fiber during degradation, and studied the influence of proportion of PGA and PLA fiber on the performance of braided yarns during degradation. The results showed that PGA fiber and PGA braided yarn had the same trend during degradation, and the degradation could be divided into three stages named as strength decrease stage, mass loss of amorphous region stage and mass loss of crystallinity region stage. In first stage, the strength remaining of fiber in braided yarn was higher than unbraided fiber, and the crystallinity decreased more obviously. In second stage, the mass loss rate of unbraided fiber was higher than fiber in braided yarn, and the crystallinity increased faster. In third stage, the crystallinity of unbraided fiber was nearly same as braided yarn, but the mass loss rate of braided yarn was faster. The results of the degradation test of 4 kinds of braided yarn with different proportion of PGA and PLA fiber indicated that with increase of PGA fiber component, the degradation rate of braided yarn increased.
This paper prepared the reinforcement of scaffold for tendon tissue engineering on the circular and flat knitting machine respectively, and discussed the influence of knitting parameters on the geometric shape and mechanical property of the reinforcement. In the process of preparation of the reinforcement on the circular knitting machine, sinking depth, drawing force and feeding tension had the influence on the geometric shape and mechanical property. The fabric prepared on smaller sinking depth, smaller drawing force and larger feeding tension, possessed the smaller stitch length, the regular stitch shape, the closer structure and higher strength. By contraries, the fabric prepared on larger sinking depth, larger drawing force and smaller feeding tension possessed the larger stitch length; the more loosen structure and lower strength. In the process of preparation of the reinforcement on the flat knitting machine, the influence of sinking depth, drawing force and feeding tension on the geometric shape and mechanical property was the same as on the circular knitting machine. The diameter of the reinforcement could be controlled by changing the number of knitting needles on the flat knitting machine, which was suitable for preparation of the reinforcement that had specific geometric requirement.
This paper studied the performance during degradation in vitro and cell attachment of the reinforcement of scaffold for tendon tissue engineering, and set up a geometric model of a small-diameter circular plain knitted fabric. The influence of proportion of PGA and PLA fiber on the performance during degradation in vitro and cell attachment was studied. The results showed that with the increase of the PGA fiber component, the degradation rate of the reinforcement of scaffold increase, and cells attached on the reinforcement better. A geometric model of a small-diameter circular plain knitted fabric was set up to analyze the geometric shape of the stitch. The geometric characteristics under extreme condition for both transverse and longitudinal extension were given. The formula for calculating surface porosity was developed.
This paper prepared an integrated scaffold for tendon tissue engineering, and studied its properties. The results showed that the scaffold for tendon tissue engineering with a "core-sheath" structure possessed reasonable geometric shape, high porousity and excellent mechanical performance. The degradation could be divided into three stages. The main characteristic of the first stage was the sharp decrease of strength, which could be named as "strength decrease stage". The main characteristic of the second stage was the sharp increase of mass loss, which could be named as "mass loss stage". The mass loss and strength were stable in third stage which could be named as "quasi-stable stage".
This paper constructed a tissue engineered tendon by cell-seeding on the scaffold, and evaluated the feasibility of clinical application of the scaffold. The maximum tensile load of the cell-scaffold was 63N. The surface of the cell-scaffold was smooth, and the reinforcement and PGA fibers combined closely. The geometric shape of the cell-scaffold changed obviously during construction. The diameter became smaller, and the length became longer. Cells attached well both on the reinforcement and PGA fibers, and produced a lot of extracellular matrices. The tissue constructed using the scaffold for tendon tissue engineering with a "core-sheath" structure, possessed the close structure, the arrangement of the collagen along the mechanical axis direction, and the similar proportion of cell/ collagen with a normal tendon. The scaffold was feasible for the clinical application.
本文根据组织工程肌腱支架的设计原则,选用PGA纤维和PLA纤维作为支架材料,设计了一种将两种纤维联合使用、具有“芯-鞘”结构的新型组织工程肌腱支架,“芯”为细胞的黏附和增殖提供场所,而“鞘”是支架增强体,在组织形成前提供足够的力学强度。其中“芯”为PGA纤维须条,“鞘”为小口径管状纬平针织物。制备支架增强体的纱线采用由PGA纤维和PLA纤维组成的编织线。
本文对组织工程肌腱支架材料使用的PGA纤维和PLA纤维的基本性能、体外降解性能、细胞黏附性能进行了较为系统、全面的研究,并探讨了熔融纺丝工艺参数对PGA纤维降解性能的影响。本文研究表明,PGA纤维与PLA纤维具有良好的力学性能,可以在纺织技术中应用。PGA纤维的降解速度快于PLA纤维,在降解8周后失去几乎全部质量,在降解2周后失去绝大部分强力,在整个降解过程中表面形态发生了明显的变化,结晶度的上升说明了纤维的非结晶区降解速度较快。而PLA纤维在整个降解过程中的变化不明显。细胞在PGA纤维上的黏附要好于在PLA纤维上的黏附,并分泌了大量的细胞外基质。纺丝过程中牵伸倍数、牵伸温度和纺丝所用聚合物的特性粘度因素对PGA纤维的降解性能有着重要的影响。在实验范围内,较高牵伸倍数条件下制得的PGA纤维在降解过程中的降解速率较慢；较高牵伸温度条件下得到的PGA纤维降解速率较快；特性粘度较高的聚合物在相同纺丝条件下制得的纤维比由较低特性粘度的聚合物制得的纤维降解速率慢。
本文研究了组织工程肌腱支架增强体用编织线的制备过程,探讨了编织加工对PGA纤维降解性能的影响,还研究了编织线中不同比例的PGA纤维和PLA纤维组分对编织线降解性能的影响。研究表明,PGA纤维和PGA编织线的降解大体趋势相同,两者的降解过程均可分为“强力下降期”、“非结晶区质量损失期”、“结晶区质量损失期”三个阶段。在第一阶段,编织线中的纤维断裂强力保持率始终高于未经编织的纤维。在第二阶段,未经编织的纤维的质量损失率始终高于编织线,结晶度的增大速率也快于编织线。在第三阶段,未经编织的纤维和编织线的结晶度趋于一致,但编织线的质量损失速率较大。对4种PGA与PLA纤维成分比例不同的编织线进行体外降解试验表明,降解过程中随着编织线中PGA纤维成分比例的增大,编织线的降解速度也不断加快。
本文分别在圆机和横机上制备了作为组织工程肌腱增强体的小口径管状纬平针织物,并探讨了针织工艺参数对其几何形状、力学性能的影响。在用圆机制备组织工程肌腱增强体的过程中,弯纱深度、牵拉力和给纱张力等工艺参数对织物的几何形态以及力学性能产生影响。在弯纱深度较小、牵拉力较小以及给纱张力较大的条件下制得的织物线圈长度较小,线圈比较规整,歪斜少,结构较为紧密,断裂强力较高；而在弯纱深度较大、牵拉力较大以及给纱张力较小的条件下制得的织物线圈长度较大,结构较为稀松,断裂强力较低。在用横机制备组织工程肌腱增强体的过程中,弯纱深度、牵拉力和给纱张力等工艺参数对织物的几何形态以及力学性能的影响与在小口径圆机上一致。可通过改变横机编织针数的方法控制支架增强体直径,适用于有明确几何形态要求的组织工程肌腱支架增强体的制备。
本文对组织工程肌腱支架增强体的体外降解性能和细胞黏附性能进行了研究,建立了小口径管状纬平针织物的几何模型。研究了不同PGA和PLA纤维成分比例对组织工程肌腱支架增强体的体外降解性能和细胞黏附性能的影响,发现在支架增强体中PGA纤维成分越多,降解速率越快,在降解过程中力学性能下降程度越大,细胞在支架增强体上的黏附情况越好,优选4PGA/2PLA编织线制备支架增强体。本文建立了小口径管状纬平针织物的几何模型,对线圈的几何形态进行了分析,给出了线圈横向和纵向变形达到极限时的几何特点,并推导了表面孔隙率的计算公式。
本文制备了完整的组织工程肌腱支架,并对完整支架的性能进行了测试和研究。研究表明该“芯-鞘”结构的组织工程肌腱支架具有合适的几何形状,较高的孔隙率以及良好的力学性能。“芯-鞘”结构的组织工程肌腱支架的整个降解过程分为三个阶段,第一阶段的主要特点为强力的大幅下降,可称之为“强力下降期”;第二阶段的主要特点为质量损失率大幅上升,可称之为“质量损失期”；第三阶段,组织工程肌腱支架的质量损失率和强力保持率均较为稳定,可称之为“准稳定期”。
本文在组织工程肌腱支架上接种细胞后进行体外构建,对该支架应用于临床的可能性进行评价。构建一周后的细胞-支架复合体的断裂强力为63N；细胞-支架复合物的表面较为光滑,表层的支架增强体与中心部分的PGA纤维须条结合紧密,细胞-支架复合物的几何形状在构建过程中发生了明显的变化,其直径变细,而长度变长；细胞在支架增强体和PGA纤维须条上均黏附较好,而且分泌了大量的细胞外基质。利用该种具有“芯-鞘”结构的组织工程肌腱支架构建出的组织结构致密,胶原沿力学轴向排列,细胞／胶原比与正常肌腱近似,具有临床应用的可能性。
Injures to tendons are very common in the physically active population. However, some limitations exist in the present therapies. The tissue engineering keeps the promise for tendon repair. Using the tissue engineering for tendon repair is as follows: combined the expanded cells and a biodegradable scaffold to form a composite, and cells proliferate, differentiate and produce the extracellular matrices to form a new tissue after grafting it in the injury position. The scaffold plays a key role in tissue engineering. It not only plays a support role to keep the original tissue shape, but also plays a template role to provide a place for cell attachment, growth, proliferation and differentiation. The scaffold which can conduct the tissue regeneration and control the structure of the tissue is a key factor to determine whether the tissue engineering can be used in clinical treatment or not. The present scaffold for tendon tissue engineering possessed the good biocompatibility, and cells could attach, proliferate and produce extracellular matrices. However, those scaffolds could not meet the mechanical requirements during degradation. This paper designed and prepared a novel scaffold for tendon tissue engineering. It not only could provide a place for cell attachment, proliferation and producing extracellular matrices, but also could meet the mechanical requirements during degradation.
This paper designed a novel scaffold for the tendon tissue engineering according to the design principles of tissue engineering scaffolds. The scaffold composed of polyglycolide (PGA) and polylactide (PLA) fibers possessed a "core-sheath" structure. The "core" provided a place for cell attachment and proliferation, and the "sheath" was a scaffold reinforcement, which provided sufficient strength before tissue formation. The "core" was composed of PGA fibers, and the "sheath" was a small-diameter circular plain knitted fabric which is made from PGA/PLA braided yarns.
This paper studied the basic properties, performance during degradation in vitro, and cell-fiber reaction of PGA and PLA fiber. The results showed that both PGA and PLA fiber possessed the good mechanical properties, and could be used in textile technologies. The PGA fiber degraded faster than PLA fiber. It lost almost all the mass after 8-week degradation, but lost almost all the tensile strength after 2-week degradation. The morphology changed obviously during degradation. The crystallinity increased firstly and then decreased with the degradation time, which indicated the degradation occurred in the amorphous region. However, the PLA fiber changed not obviously during degradation. Cells attached on PGA fibers much better than on PLA fibers, and cells on PGA fibers produced more matrix. Drawing multiple, drawing temperature and inherent viscosity of polymer had the influence on the performance of PGA fiber during degradation. The PGA fiber produced on higher drawing multiple degraded more slowly. The PGA fiber produced on higher drawing temperature degraded faster. The PGA fiber made from higher inherent viscosity polymer degraded more slowly.
This paper studied the preparation of the braided yarns used in the reinforcement of scaffold for tendon tissue engineering, discussed the influence of braiding process on the performance of PGA fiber during degradation, and studied the influence of proportion of PGA and PLA fiber on the performance of braided yarns during degradation. The results showed that PGA fiber and PGA braided yarn had the same trend during degradation, and the degradation could be divided into three stages named as strength decrease stage, mass loss of amorphous region stage and mass loss of crystallinity region stage. In first stage, the strength remaining of fiber in braided yarn was higher than unbraided fiber, and the crystallinity decreased more obviously. In second stage, the mass loss rate of unbraided fiber was higher than fiber in braided yarn, and the crystallinity increased faster. In third stage, the crystallinity of unbraided fiber was nearly same as braided yarn, but the mass loss rate of braided yarn was faster. The results of the degradation test of 4 kinds of braided yarn with different proportion of PGA and PLA fiber indicated that with increase of PGA fiber component, the degradation rate of braided yarn increased.
This paper prepared the reinforcement of scaffold for tendon tissue engineering on the circular and flat knitting machine respectively, and discussed the influence of knitting parameters on the geometric shape and mechanical property of the reinforcement. In the process of preparation of the reinforcement on the circular knitting machine, sinking depth, drawing force and feeding tension had the influence on the geometric shape and mechanical property. The fabric prepared on smaller sinking depth, smaller drawing force and larger feeding tension, possessed the smaller stitch length, the regular stitch shape, the closer structure and higher strength. By contraries, the fabric prepared on larger sinking depth, larger drawing force and smaller feeding tension possessed the larger stitch length; the more loosen structure and lower strength. In the process of preparation of the reinforcement on the flat knitting machine, the influence of sinking depth, drawing force and feeding tension on the geometric shape and mechanical property was the same as on the circular knitting machine. The diameter of the reinforcement could be controlled by changing the number of knitting needles on the flat knitting machine, which was suitable for preparation of the reinforcement that had specific geometric requirement.
This paper studied the performance during degradation in vitro and cell attachment of the reinforcement of scaffold for tendon tissue engineering, and set up a geometric model of a small-diameter circular plain knitted fabric. The influence of proportion of PGA and PLA fiber on the performance during degradation in vitro and cell attachment was studied. The results showed that with the increase of the PGA fiber component, the degradation rate of the reinforcement of scaffold increase, and cells attached on the reinforcement better. A geometric model of a small-diameter circular plain knitted fabric was set up to analyze the geometric shape of the stitch. The geometric characteristics under extreme condition for both transverse and longitudinal extension were given. The formula for calculating surface porosity was developed.
This paper prepared an integrated scaffold for tendon tissue engineering, and studied its properties. The results showed that the scaffold for tendon tissue engineering with a "core-sheath" structure possessed reasonable geometric shape, high porousity and excellent mechanical performance. The degradation could be divided into three stages. The main characteristic of the first stage was the sharp decrease of strength, which could be named as "strength decrease stage". The main characteristic of the second stage was the sharp increase of mass loss, which could be named as "mass loss stage". The mass loss and strength were stable in third stage which could be named as "quasi-stable stage".
This paper constructed a tissue engineered tendon by cell-seeding on the scaffold, and evaluated the feasibility of clinical application of the scaffold. The maximum tensile load of the cell-scaffold was 63N. The surface of the cell-scaffold was smooth, and the reinforcement and PGA fibers combined closely. The geometric shape of the cell-scaffold changed obviously during construction. The diameter became smaller, and the length became longer. Cells attached well both on the reinforcement and PGA fibers, and produced a lot of extracellular matrices. The tissue constructed using the scaffold for tendon tissue engineering with a "core-sheath" structure, possessed the close structure, the arrangement of the collagen along the mechanical axis direction, and the similar proportion of cell/ collagen with a normal tendon. The scaffold was feasible for the clinical application.
引文
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