CN104606713B - Three parallel collagen fibers - silk scaffold and preparation method and application - Google Patents

Three parallel collagen fibers - silk scaffold and preparation method and application Download PDF

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CN104606713B
CN104606713B CN201510005188.3A CN201510005188A CN104606713B CN 104606713 B CN104606713 B CN 104606713B CN 201510005188 A CN201510005188 A CN 201510005188A CN 104606713 B CN104606713 B CN 104606713B
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silk
collagen fibers
parallel
dimensional
collagen
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CN104606713A (en
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欧阳宏伟
沈炜亮
郑泽峰
陈晓
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浙江大学
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Abstract

本发明提供了一种三维平行胶原纤维-蚕丝支架及其制备方法,所述支架由编织蚕丝基架和包覆在编织蚕丝基架上、下方表面的平行胶原纤维层构成,所述的编织蚕丝基架是由蚕丝编织成网孔大小为0.25~25mm2的网片结构,所述平行胶原纤维的孔隙直径大小为10~300um,所述的编织蚕丝是去除了丝胶蛋白的编织蚕丝;所述支架可作为生物支架应用于肌腱/韧带的修复;本发明三维平行胶原纤维-蚕丝支架制作方法简单,生物相容性好,力学性能佳,支架的拓扑结构与肌腱/韧带相似,能够促进其表面生长的干细胞向肌腱/韧带分化,促进肌腱/韧带再生。 The present invention provides a three-dimensional parallel collagen fibers - silk stent and its preparation method, the stent is made of braided silk fibroin coated carrier and silkworm fibroin weaving rack, parallel collagen fibers beneath the surface layer, the braided silk base frame is made of silk woven mesh size of 0.25 ~ 25mm2 mesh structure of the collagen fibers parallel pore diameter of 10 ~ 300um, the braided silk braided silk is sericin to the addition; the stent may be applied to the tendon / ligament as a biological scaffold; three parallel collagen fibers of the present invention - silk holder manufacturing method is simple, good biocompatibility, good mechanical properties, similar topology and tendon / ligament scaffold, the surface can promote growth of stem cells into the tendon / ligament, promote tendon / ligament regeneration.

Description

三维平行胶原纤维一蚕丝支架及其制备方法与应用 A three-dimensional parallel collagen fibers silk scaffold and preparation method and application

(一) (One)

技术领域 FIELD

[0001] 本发明涉及一种用于肌腱/韧带修复的三维的支架材料及其制备方法,特别涉及一种三维平行胶原纤维一蚕丝支架及其制备方法和应用。 [0001] The present invention relates to a three-dimensional scaffold and preparation method tendon / ligament repair is used, in particular, relates to a three-dimensional silk scaffold parallel collagen fibers and a preparation method and application.

(二) (two)

背景技术 Background technique

[0002]随着我国经济的发展和人民生活水平的提高,人们的健康观念也在提升,出于对健康体魄的向往,国人参加体育锻炼和竞赛活动的数量明显增加,运动损伤也越来越多,其中韧带/肌腱损伤占50 %以上。 [0002] As China's economic development and people's living standards, people's attitudes are health promotion, out of longing for good health, the number of people to participate in physical training and competition activities increased significantly, more and more sports injuries and more, where the ligament / tendon injuries account for more than 50%. 有数据表明,每2亿人口中一年至少有上千万的肌腱损伤病例。 Data show that every 200 million people in at least tendon injury cases tens of millions a year.

[0003]当前,临床上主要靠直接缝合、自体移植、同种异体移植、异种移植和假体材料修复术来修复韧带和肌腱损伤。 [0003] Currently, clinical mainly by direct suture, autograft, allograft, xenograft prostheses and prosthetic materials to repair ligament and tendon injuries. 然而,这些手术重建再生技术都有其固有的缺陷,例如复发性撕裂、供体缺乏、供体部位并发症、免疫排斥和较差的支架整合性。 However, these surgical reconstruction regeneration techniques have inherent drawbacks such as recurrent tear, lack of donors, donor site complications, immune rejection and poor integration stent.

[0004]目前临床方法的缺点促使肌腱/韧带修复的研究转向可降解生物材料和组织工程方法。 [0004] disadvantages of current clinical methods to promote tendon / ligament repair steering research biodegradable materials and tissue engineering. 有研究者使用高分子材料纤维或者蚕丝组成辫状支架用于修复损伤肌腱,然而,这些辫状支架没有内部空隙,不能承载大量细胞,也不能让机体形成相互连接有功能的新组织。 Some researchers use a polymer material composed of fibers or silk braided scaffold for repairing damaged tendons, however, these braided stents have no internal voids, can not carry a large number of cells, or to allow the formation of new body tissues interconnected functional. 另外有研究者采用胶原接种大量细胞用于肌腱/韧带组织工程。 Another researchers use a large number of cells seeded collagen tendon / ligament tissue engineering. 这些支架解决了细胞组织容纳问题,但它们的力学性能很差,不能承受机体的生理机械力,难以应用于临床。 These scaffolds tissue receiving solve the problem, but they have a poor mechanical properties, can not withstand the mechanical forces the body's physiological and difficult to apply clinically.

[0005] 之前有研究把蚕丝和胶原海绵复合在一起,构建出了一个实用的肌腱/韧带组织工程支架。 Studies have silk and the collagen sponge together to build a practical tendon / ligament tissue engineering scaffold before [0005]. 胶原海绵提供修复细胞粘附和长入的空间,编织状蚕丝纤维提供足够的力学强度。 Collagen sponge provides repair cell adhesion and ingrowth space, braided silk fibers provide sufficient mechanical strength. 然而其在结构排列上与肌腱/韧带组织的形状相差很大,修复肌腱/韧带的结构和功能与正常肌腱/韧带相比较,也是相差甚远。 However, the shape of the structural arrangement in which the tendon / ligament tissue is very different, the structure and function to repair tendon / ligament compared to normal tendon / ligament, is very different. 一种理想的、能吸引目的细胞在损伤部位集聚并能有效引导细胞迀徙的有生物活性的蚕丝支架的组合仍然面临着巨大的挑战。 An ideal that can attract the desired cell concentration and can effectively guide the migration of cells Gan has a combination of bioactive silk scaffold still faces enormous challenges in the site of injury.

(三) (three)

发明内容 SUMMARY

[0006] 本发明目的是提供一种三维平行胶原纤维一蚕丝支架及其制备方法与应用,本发明支架具有良好的力学性能,能够提供修复细胞粘附和长入的空间,具有与肌腱/韧带相似的结构,能有效引导细胞迀徙和排列,可作为生物支架应用于肌腱/韧带的修复。 [0006] The object of the present invention to provide a three-dimensional fibrous scaffold and a silk parallel collagen preparation method and application, the stent of the present invention has good mechanical properties, can be provided to repair cell adhesion and ingrowth space, having tendon / ligament similar structure, can effectively guide Gan cell migration and arrangement can be applied to the tendon / ligament as a biological scaffold.

[0007] 本发明采用的技术方案是: [0007] aspect of the present invention is that:

[0008] 一种三维平行胶原纤维一蚕丝支架,所述支架由编织蚕丝基架和包覆在编织蚕丝基架上、下方表面的平行胶原纤维层构成,所述的编织蚕丝基架是由蚕丝编织成网孔大小为0.25〜25mm2的网片结构,所述平行胶原纤维的孔隙直径大小为10〜300um;所述的编织蚕丝是去除了丝胶蛋白的编织蚕丝。 [0008] A three-dimensional collagen fibers parallel to a silk stent, the stent woven silk fibroin in a rack, parallel collagen fibers below the surface layers composed of woven silk fibroin-coated carrier and said carrier is a woven silk fibroin silk woven into a mesh size of the mesh structure 0.25~25mm2, said parallel collagen fibers pore diameter of 10~300um; the silk weave is removed woven silk sericin.

[0009] 本发明三维平行胶原纤维一蚕丝支架,优选所述编织蚕丝基架的网孔大小为I〜9mm2 ο [0009] The present invention is a three-dimensional parallel collagen fibers a silk scaffold, preferably the braided mesh silkworm silk-frame size I~9mm2 ο

[0010] 优选所述平行胶原纤维的孔隙直径大小为50〜150um。 [0010] Preferably, the pore diameter of collagen fibers is parallel 50~150um.

[0011] 本发明还提供了一种所述三维平行胶原纤维一蚕丝支架的制备方法,所述的制备方法包括如下步骤: [0011] The present invention further provides a method of preparing a three-dimensional parallel to the collagen fiber a silk scaffold, prepared according to the method comprising the steps of:

[0012] (I)将编织蚕丝基架去除丝胶蛋白; [0012] (I) the carrier removal braided silk fibroin sericin;

[0013] (2)在去除了丝胶蛋白的编织蚕丝基架的上方、下方分别注入浓度为5〜20mg/ml的胶原溶液,并使编织蚕丝基架两侧胶原溶液层的厚度为I〜10mm; [0013] (2) In addition to the above woven silk fibroin sericin frame below were injected at a concentration of 5~20mg / ml of collagen solution, and woven silk fibroin frame sides collagen solution layer having a thickness of I~ 10mm;

[0014] (3)低温冷却使胶原凝固,然后真空减压干燥; [0014] (3) a cryogenic cooling collagen coagulation, and then dried in vacuo under reduced pressure;

[0015] ⑷最后进行干热交联,得到所述的三维平行胶原纤维一蚕丝支架。 [0015] ⑷ final dry heat crosslinking, to obtain a three-dimensional parallel collagen fibers of the silk stent.

[0016] 本发明步骤⑴中,所述编织蚕丝基架去除丝胶蛋白的方法可以选自下列之一:碳酸钠溶液煮沸去除法、去污剂煮沸去除法、硼酸煮沸去除法或者直接加热去除法等,这些去除丝胶蛋白的方法,都适合本发明。 [0016] Step ⑴ the present invention, the braided silk fibroin sericin removal frame may be selected from one of the following methods: Method sodium carbonate solution to boil off, the boiling detergent removal method, acid method or a direct heating boil off removal method, these methods remove sericin, are suitable for the present invention.

[0017] 具体的,所述的碳酸钠溶液煮沸去除法为:将编织蚕丝基架浸没于0.2wt%Na2C03水溶液中,煮沸60〜150min,中间换水2〜5次,室温或干燥箱干燥,即得去除了丝胶蛋白的编织蚕丝基架。 [0017] Specifically, the sodium carbonate solution to boil off method: braided silk fibroin carrier was immersed in an aqueous solution of 0.2wt% Na2C03, boiled 60~150min, intermediate 2 ~ 5 times changing the water, at room temperature or drying oven, that was removed woven silk fibroin sericin frame.

[0018]具体的,所述步骤(2)按如下步骤进行:首先,在一端嵌入有温度传递金属条的模具中注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平后在溶液上表面平放去除了丝胶蛋白的编织蚕丝基架,然后在编织蚕丝基架上方注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10_,铺平即可;所述的温度传递金属条选自铝条、不锈钢条或铜条等传递温度性能好的材料,所述模具的材质选自亚克力、陶瓷或硅胶等导热性能差的材料。 [0018] Specifically, the step (2) as follows: First, a mold temperature transmitting end is embedded in the metal strip of implant concentration 5~20mg / ml of collagen solution and the solution having a thickness of I~10mm after removing paved surface flat woven silk fibroin sericin frame, and then knitted on silkworm fibroin solution square frame of implant concentration 5~20mg / ml of collagen solution and the solution having a thickness of I~10_, shop flat can; the temperature of the temperature transfer properties of the metal strip passing good material selected aluminum, copper, stainless steel strips, or strips, a difference between the mold material is selected from acrylic material, ceramic or other thermal conductivity of silica gel.

[0019] 本发明步骤⑶中,使胶原凝固的低温冷却方法可以采用下列之一:装有液氮的泡沫盒、制冷机或低温冰块等,这些制造温度梯度且使胶原凝固的方法,都适合本发明。 [0019] Step ⑶ the present invention, the solidified collagen cryogenic cooling method may be one of the following: a foam box with liquid nitrogen, or low temperature refrigerator ice etc. These temperature gradients and a method for producing collagen coagulation, are suitable for the present invention.

[0020]具体推荐本发明三维平行胶原纤维一蚕丝支架按如下步骤制备: [0020] DETAILED recommended a three-dimensional silk scaffold parallel collagen fibers of the present invention was prepared as follows:

[0021] (I)将编织蚕丝基架浸没于0.2wt%Na2C03水溶液中,煮沸60〜150min,中间换水2〜5次,室温或干燥箱干燥,得到去除了丝胶蛋白的编织蚕丝基架; [0021] (I) braided silk fibroin carrier was immersed in an aqueous solution of 0.2wt% Na2C03, boiled 60~150min, intermediate 2 ~ 5 times changing the water, at room temperature or a drying oven, to obtain silk fibroin to weave frame except sericin ;

[0022] (2)在一端嵌入有铝条的亚克力模具中注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平后在溶液上表面平放步骤⑴所得去除了丝胶蛋白的编织蚕丝基架,然后在编织蚕丝基架上方注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平; [0022] (2) at one end is embedded in the aluminum mold acrylic implant concentration of 5~20mg / ml of collagen solution and the solution having a thickness of I~10mm, flat surface after paving step on the resulting solution was removed ⑴ braided silk fibroin sericin frame, then frame side woven silk fibroin concentration of implanted 5~20mg / ml of collagen solution and the solution having a thickness of I~10mm, paving;

[0023] (3)将亚克力模具的另一端与装有液氮的泡沫盒相连,调整泡沫盒与模具的距离为10〜100mm,待胶原全部凝固后,置于真空抽干机中抽干; [0023] (3) is connected to the other end of the mold acrylic foam cartridges containing liquid nitrogen, the mold box and the foam adjusting a distance 10~100mm, until all of the solidified collagen, sucked dry in a vacuum dryer drained;

[0024] ⑷最后进行干热交联,方法为:在〈lOmbar的真空干燥箱中,22°C放置一天,110°C放置三天,65 °C放置一天,即得所述的三维平行胶原纤维一蚕丝支架。 [0024] ⑷ final dry heat crosslinking method: In <lOmbar in a vacuum oven, 22 ° C for one day, 110 ° C placing three days, 65 ° C for one day, to obtain the three-dimensional parallel collagen a silk fiber holder.

[0025] 本发明三维平行胶原纤维一蚕丝支架可作为生物支架应用于肌腱/韧带的修复。 [0025] The three-dimensional parallel collagen fibers of the invention may be applied as a silk scaffold bioscaffolds the tendon / ligament.

[0026]与现有技术相比,本发明的有益效果主要体现在: [0026] Compared with the prior art, the beneficial effects of the present invention is mainly embodied in:

[0027] 1、本发明三维平行胶原纤维一蚕丝支架制作方法简单,其形状可以根据模具控制调整,其孔隙可以通过温度及胶原浓度调整; [0027] 1, the present invention is a three-dimensional parallel collagen fibers silk holder manufacturing method is simple, the shape of the mold can control adjustment, which can be adjusted by the aperture collagen concentration and temperature;

[0028] 2、本发明三维平行胶原纤维一蚕丝支架生物相容性好,胶原和蚕丝均可在生物体内被降解; [0028] 2, the present invention is a three-dimensional parallel collagen fibers silk scaffold good biocompatibility, silk, and collagen may be degraded in the living body;

[0029] 3、本发明三维平行胶原纤维一蚕丝支架力学性能佳,可以满足肌腱/韧带所需的强大力学; [0029] 3, the present invention is a three-dimensional parallel collagen fibers silk scaffold good mechanical properties, the mechanical power needed to meet the tendon / ligament;

[0030] 4、本发明三维平行胶原纤维一蚕丝支架的拓扑结构与肌腱/韧带相似,能够促进其表面生长的干细胞向肌腱/韧带分化,促进肌腱/韧带再生。 [0030] 4, similar topology and tendon / ligament-dimensional parallel collagen fibers of the present invention, a silk scaffold, a surface capable of promoting the growth of stem cells into the tendon / ligament, promote tendon / ligament regeneration.

(四) (four)

附图说明 BRIEF DESCRIPTION

[0031]图1:编织蚕丝大体图片; [0031] FIG 1: a generally braided silk image;

[0032]图2:三维平行胶原纤维一蚕丝支架大体图; [0032] Figure 2: a three-dimensional parallel collagen fibers substantially FIG silk scaffold;

[0033]图3:平行胶原纤维电镜扫描图,图3-1为6mg/ml胶原,图3-2为10mg/ml胶原,图3-3为18mg/ml胶原; [0033] Figure 3: SEM of parallel collagen fibers, and FIG. 3-1 is a 6mg / ml collagen, FIG. 3-2 is a 10mg / ml collagen, FIG. 3-3 is a 18mg / ml collagen;

[0034]图4:肌腱干细胞/前体细胞在三维平行胶原纤维一蚕丝支架上的增殖; [0034] FIG. 4: tendon stem cell precursor cell proliferation / parallel collagen fibers in a three-dimensional silk stent;

[0035]图5:兔子肩袖损伤修复4周后HE染色,A为紊乱胶原纤维一蚕丝支架组,B为三维平行胶原纤维一蚕丝支架组; [0035] FIG. 5: rabbit rotator cuff repair 4 weeks after HE staining, A is a disordered collagen silk stent group, B is a three-dimensional parallel collagen fibers silk stent;

[0036]图6:兔子肩袖损伤修复4周后Masson染色,A为紊乱胶原纤维一蚕丝支架组,B为三维平行胶原纤维一蚕丝支架组; [0036] FIG. 6: rabbit rotator cuff repair Masson staining after four weeks, A is a disordered collagen silk stent group, B is a three-dimensional parallel collagen fibers silk stent;

[0037]图7:兔子肩袖损伤修复4周后新生肌腱胶原含量; [0037] Figure 7: newborn rabbit rotator cuff repair tendon collagen content after 4 weeks;

[0038]图8:兔子肩袖损伤修复4周后肌腱相关基因表达。 [0038] Figure 8: rabbit rotator cuff tendon repair related gene expression after 4 weeks.

(五) (Fives)

具体实施方式 Detailed ways

[0039]下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此: [0039] The following embodiments in conjunction with specific embodiments of the present invention is further described, although the scope of the present invention is not limited thereto:

[0040] 实施例1 [0040] Example 1

[0041] 将网孔大小为Imm2的编织蚕丝去除丝胶蛋白,剪成2*2cm (见图1)。 [0041] The mesh size to remove sericin Imm2 braided silk, cut into 2 * 2cm (see FIG. 1). 在编织蚕丝下方注入2mm厚的6mg/ml的胶原,在编织蚕丝上方注入3mm厚的6mg/ml的胶原。 2mm thick collagen injection 6mg / ml below the braided silk, collagen injection 3mm thick 6mg / ml above the silk braid. 调整装液氮的泡沫盒与模具的距离为5cm。 Adjusting means of liquid nitrogen from the mold box and the foam was 5cm. 凝固后置于真空抽干机中抽干,然后进行干热交联,得到孔隙直径大小约200um的三维平行胶原纤维一蚕丝支架。 After solidification in a vacuum dryer drained drained, and then subjected to dry thermal crosslinking, to obtain a three-dimensional pore diameter parallel collagen fibers in a silk scaffold about 200um. 观察其电镜扫描图片(见图3-1)。 Observed SEM image (see FIG. 3-1).

[0042] 实施例2 [0042] Example 2

[0043] 将网孔大小为Imm2的编织蚕丝去除丝胶蛋白,剪成2*2cm。 [0043] The mesh size to remove sericin Imm2 braided silk, cut into 2 * 2cm. 在编织蚕丝下方注入2mm厚的I Omg/ml的胶原,在编织蚕丝上方注入3mm厚的I Omg/ml的胶原。 2mm thick injection of collagen I Omg / ml below the braided silk, collagen injection 3mm thick I Omg / ml above the silk braid. 调整装液氮的泡沫盒与胶原、蚕丝的距离为5cm。 Adjusting means nitrogen bubble cassette collagen, silk distance of 5cm. 凝固后置于真空抽干机中抽干,然后进行干热交联,得到孔隙直径大小约SOum的三维平行胶原纤维一蚕丝支架(见图2)。 After solidification in a vacuum dryer drained drained, and then subjected to dry thermal crosslinking, to obtain pore diameter of about three-dimensional parallel collagen fibers SOum a silk scaffold (see FIG. 2). 观察其电镜扫描图片(见图3- Observed SEM image (Figure 3-2

2) ο 2) ο

[0044] 实施例3 [0044] Example 3

[0045] 将网孔大小为Imm2的编织蚕丝去除丝胶蛋白,剪成2*2cm。 [0045] The mesh size to remove sericin Imm2 braided silk, cut into 2 * 2cm. 在编织蚕丝下方注入2mm厚的18mg/ml的胶原,在编织蚕丝上方注入3mm厚的18mg/ml的胶原。 2mm thick collagen injection 18mg / ml in the lower woven silk, 3mm thick collagen injection 18mg / ml above the silk braid. 调整装液氮的泡沫盒与胶原、蚕丝的距离为5cm。 Adjusting means nitrogen bubble cassette collagen, silk distance of 5cm. 凝固后置于真空抽干机中抽干,然后进行干热交联,得到孔隙直径大小约20um的三维平行胶原纤维一蚕丝支架。 After solidification in a vacuum dryer drained drained, and then subjected to dry thermal crosslinking, to obtain a three-dimensional pore diameter parallel collagen fibers in a silk scaffold about 20um. 观察其电镜扫描图片(见图3-3)。 Observed SEM image (see FIG. 3-3).

[0046] 实施例4 [0046] Example 4

[0047] 在实施例2中所得的支架上种植肌腱干细胞/前体细胞。 [0047] Growing tendon stem / progenitor cells in the scaffold obtained in Example 2. 通过cck-8试剂盒检测1、 By cck-8 kit 1,

3、7天细胞的增殖情况(见图4),表明细胞在三维平行胶原纤维一蚕丝支架上增殖正常。 Cell proliferation 3,7 days (see Figure 4), indicating that the proliferation of normal cells in a three-dimensional silk scaffold parallel collagen fibers.

[0048] 实施例5 [0048] Example 5

[0049] 三维平行胶原纤维一蚕丝支架在肩袖损伤中的应用。 Application of [0049] a three-dimensional silk scaffold parallel collagen fibers in the rotator cuff injury.

[0050] (I)建立动物模型:将5只约2500g的雌性兔子,经过静脉麻醉后,切开皮肤、筋膜及部分肌肉,将R上肌肌腱充分暴露,在贴近肱骨大结节处切断R上肌肌腱。 [0050] (I) established animal model: the five female rabbits of about 2500g, after intravenous anesthesia, skin incision, fascia and part of the muscle, the tendon on the R fully exposed, cut close to the greater tuberosity of the humerus R on the tendon.

[0051] (2)支架植入:同一只兔子左右对照,实验组即实施例2中所得的支架,对照组的胶原浓度及蚕丝分布同实施例2,直接放-20 0C冰箱凝固,冻干、交联同实施例2 ο将支架一端直接与R上肌肌腱断端缝合,另一端通过骨隧道与肱骨大结节相连。 [0051] (2) stent implantation: about the same rabbits control, i.e., the experimental group stent embodiments, silk and collagen concentration distribution in the control group of Example 2 obtained in Example 2, is placed directly -20 0C refrigerator freezing, freeze-dried crosslinked with Example 2 ο end of the bracket directly to the tendon stump suturing R, and the other end connected through the greater tuberosity of humerus bone tunnel.

[0052] (3)组织修复能力:术后4周,取出兔子冈上肌肌腱,进行HE染色、Masson染色、胶原含量测定及肌腱相关基因检测。 [0052] (3) in tissue repair: After four weeks, the rabbit supraspinatus tendon removed, for HE staining, Masson staining, and measuring collagen content related tendon genetic testing.

[0053] HE染色结果:实验组支架内部也有新生肌腱长入,而对照支架内部新生肌腱长入很少(见图5)。 [0053] HE staining: the experimental group holder has internal neonatal tendon ingrowth, while the control neonatal tendon inner support ingrowth few (see FIG. 5). 结果表明三维平行胶原纤维一蚕丝支架能够促进肌腱再生。 The results show a three-dimensional parallel collagen fibers silk scaffold can promote regeneration of the tendon.

[0054] Masson染色结果:实验组支架内部的新生肌腱比对照组更多、更成熟(见图6)。 [0054] Masson staining: experimental group holder newborn inside tendons more, more mature than the control group (see FIG. 6). 结果表明三维平行胶原纤维一蚕丝支架肌腱再生的促进作用比紊乱胶原纤维一蚕丝支架强。 The results show that the promoting effect of collagen fibers parallel to a three-dimensional silk scaffold regenerated tendon collagen disorders stronger than a silk scaffold.

[0055] 胶原含量测定:实验组的胶原含量为(131.12 ± 36.73) mg/g,对照组为(121.32 土27.12)mg/g (见图7),表明三维平行胶原纤维一蚕丝支架能够促进更多的胶原生成,促进肌腱再生。 [0055] Determination of collagen: collagen content of the experimental group (131.12 ± 36.73) mg / g, the control group (121.32 soil 27.12) mg / g (see FIG. 7), parallel collagen fibers show a three-dimensional silk scaffold can promote more more collagen production, promote tendon regeneration.

[0056] 肌腱相关基因表达:0)11,0)13,1'11(3,8811均在实验组有更高的表达(见图8)。表明三维平行胶原纤维一蚕丝支架能够促进肌腱相关基因表达,从而促进肌腱再生。 [0056] tendon gene expression: 0) 11, 0) 13,1'11 (3,8811 in the experimental group were more highly expressed (see FIG. 8) of parallel collagen fibers showed a three-dimensional scaffold can promote silk related tendon. gene expression, thereby facilitating tendon regeneration.

Claims (8)

1.一种三维平行胶原纤维一蚕丝支架,其特征在于,所述支架由编织蚕丝基架和包覆在编织蚕丝基架上、下方表面的平行胶原纤维层构成,所述的编织蚕丝基架是由蚕丝编织成网孔大小为0.25〜25mm2的网片结构,所述平行胶原纤维的孔隙直径大小为10〜300um;所述的编织蚕丝是去除了丝胶蛋白的编织蚕丝; 所述的三维平行胶原纤维一蚕丝支架按如下方法制备得到: (I)将编织蚕丝基架去除丝胶蛋白; ⑵首先,在一端嵌入有温度传递金属条的模具中注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平后在溶液上表面平放去除了丝胶蛋白的编织蚕丝基架,然后在编织蚕丝基架上方注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平即可;所述的温度传递金属条选自铝条、不锈钢条或铜条,所述模具的材质选自亚克力、陶瓷或硅胶; ⑶低温冷 1. A three-dimensional collagen fibers parallel to a silk stent, wherein said stent is made of braided silk fibroin coated carrier and silkworm fibroin weaving rack, parallel collagen fibers beneath the surface layer, the braided silk fibroin frame silk is woven into the mesh size of the mesh structure 0.25~25mm2, said parallel collagen fibers pore diameter of 10~300um; the silk weave is removed woven silk sericin; the three-dimensional a parallel collagen fibers silk scaffold was prepared by the following method: (I) the carrier removal braided silk fibroin sericin; ⑵ first, there is a mold temperature at the transmitting end is embedded in the metal strip of implant concentration 5~20mg / ml of collagen solution and the solution having a thickness of I~10mm, removed flat surface after paving braided silk fibroin-sericin frame over the solution, and then woven silk fibroin side frame of implant concentration 5~20mg / ml of collagen solution, and the thickness of the solution I~10mm, can pave the way; the temperature of the metal strip is selected from aluminum transfer, stainless steel or copper strip article, the mold material is selected from acrylic, ceramic or silica; cold ⑶ 使胶原凝固,然后真空减压干燥; ⑷最后进行干热交联,得到所述的三维平行胶原纤维一蚕丝支架。 Collagen coagulation, and then dried in vacuo under reduced pressure; ⑷ finally dry heat crosslinking, to obtain a three-dimensional parallel collagen fibers of the silk stent.
2.如权利要求1所述的三维平行胶原纤维一蚕丝支架,其特征在于,所述编织蚕丝基架的网孔大小为I〜9mm2。 2. The three-dimensional collagen fibers parallel to a silk holder according to claim 1, wherein the braided mesh size of silkworm silk-frame I~9mm2.
3.如权利要求1所述的三维平行胶原纤维一蚕丝支架,其特征在于,所述平行胶原纤维的孔隙直径大小为50〜150umo The three-dimensional collagen fibers parallel to a silk holder according to claim 1, wherein said parallel collagen fibers pore diameter of 50~150umo
4.如权利要求1所述的三维平行胶原纤维一蚕丝支架,其特征在于步骤(I)中,所述编织蚕丝基架去除丝胶蛋白的方法选自下列之一:碳酸钠溶液煮沸去除法、去污剂煮沸去除法、硼酸煮沸去除法或者直接加热去除法。 A method of three-dimensional parallel collagen fibers of the silk stent as claimed in claim 1, wherein the step (I), the braided silk fibroin-sericin is removed carrier is selected from one of the following: sodium carbonate solution was boiled removal method , boiling detergent removal method, acid method or a direct heating boil off removal method.
5.如权利要求4所述的三维平行胶原纤维一蚕丝支架,其特征在于,所述的碳酸钠溶液煮沸去除法为:将编织蚕丝基架浸没于0.2wt ^Na2CO3水溶液中,煮沸60〜150min,中间换水2〜5次,室温或干燥箱干燥,即得去除了丝胶蛋白的编织蚕丝基架。 The three-dimensional parallel collagen fibers a silk holder according to claim 4, wherein said sodium carbonate solution to boil off method: braided silk fibroin in an aqueous carrier ^ Na2CO3 0.2wt immersed in boiling 60~150min intermediate 2 ~ 5 times changing the water, at room temperature or a drying oven, to obtain silk fibroin removed woven carrier sericin.
6.如权利要求1所述的三维平行胶原纤维一蚕丝支架,其特征在于所述步骤(3)中,使胶原凝固的低温冷却方法采用下列之一:装有液氮的泡沫盒、制冷机或低温冰块。 6. The three-dimensional collagen fibers parallel to a silk holder according to claim 1, wherein said step (3) in the solidified collagen cryogenic cooling method using one of the following: a foam box with liquid nitrogen, refrigerator or low temperature ice.
7.如权利要求1所述的三维平行胶原纤维一蚕丝支架,其特征在于,所述的三维平行胶原纤维一蚕丝支架按如下方法制备得到: (1)将编织蚕丝基架浸没于0.2wt%Na2C03水溶液中,煮沸60〜150min,中间换水2〜5次,室温或干燥箱干燥,得到去除了丝胶蛋白的编织蚕丝基架; (2)在一端嵌入有铝条的亚克力模具中注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平后在溶液上表面平放步骤(I)所得去除了丝胶蛋白的编织蚕丝基架,然后在编织蚕丝基架上方注入浓度为5〜20mg/ml的胶原溶液,并使溶液厚度为I〜10mm,铺平; (3)将亚克力模具的另一端与装有液氮的泡沫盒相连,调整泡沫盒与模具的距离为10〜100mm,待胶原全部凝固后,置于真空抽干机中抽干; ⑷最后进行干热交联,方法为:在〈1mbar的真空干燥箱中,22°C放置一天,110°C放置三天,65°C放置一天,即 7. The three-dimensional collagen fibers parallel to a silk holder according to claim 1, wherein said three-dimensional collagen fibers parallel to a silk scaffolds prepared as follows: (1) braided silk fibroin carrier was immersed in 0.2wt% Na2C03 aqueous solution, boiled 60~150min, intermediate 2 ~ 5 times changing the water, at room temperature or drying oven, removed braided silk fibroin obtained sericin carrier protein; (2) embedded at one end in the aluminum mold acrylic implant concentration collagen solution 5~20mg / ml of the solution and thickness I~10mm, flat surface of step (I) in the resulting solution was removed paving braided silk fibroin sericin frame, then silkworm silk-weaving frame the top of implant concentration 5~20mg / ml of collagen solution and the solution having a thickness of I~10mm, paving; (3) is connected to the other end of the mold acrylic foam cartridges containing liquid nitrogen, the adjustment of the mold box foam distance 10~100mm, until all of the solidified collagen, sucked dry in a vacuum dryer drained; dry heat crosslinking ⑷ Finally, the method is: in <1mbar in a vacuum oven, 22 ° C for one day, 110 ° C placed three days, 65 ° C for one day, i.e., 得所述的三维平行胶原纤维一蚕丝支架。 The obtained three-dimensional collagen fibers parallel to a silk scaffold.
8.如权利要求1〜7之一所述的三维平行胶原纤维一蚕丝支架作为生物支架应用于肌腱/韧带的修复。 A three-dimensional parallel collagen fibers silk scaffold as claimed in one of claims 1~7 applied as a biological scaffold to repair tendon / ligament.
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