技术领域technical field
本发明涉及高分子材料和组织工程技术领域，具体地说，是一种新型的脂肪干细胞(Adipose stem cells,ASCs)结合POC-PLA(聚柠檬酸酯-聚乳酸)电纺丝生物补片，修复缺损组织的新方法，及其将该补片和方法在修复胸壁全层缺损中的研究应用。The invention relates to the technical field of polymer materials and tissue engineering, in particular to a new type of adipose stem cells (Adipose stem cells, ASCs) combined with POC-PLA (polycitrate-polylactic acid) electrospinning biological patch, A new method for repairing defective tissue, and the research and application of the patch and method in repairing full-thickness chest wall defects.
背景技术Background technique
大面积的全层胸壁缺损重建仍然是困扰整形科、心胸外科的难题，临床上此类手术往往需要心胸外科医生重建封闭胸腔，重建骨性支架，同时软组织的缺损则需要整形外科医生灵活运用合适的皮瓣进行移植修复。目前影响胸壁缺损重建效果的因素还是在于合适的选择胸壁重建的材料。虽然目前临床上有钛板、人工骨、各种补片可供选择，但是尚无一种能完全达到胸壁重建的理想要求。临床前研究正不停寻找新的更接近正常胸壁形态与功能的材料，随着材料学和组织工程学的发展，一种更合适的重建材料势必将更显著地改善此类患者的术后生存质量。一种理想的胸壁替代材料需要有极佳的抗张力和弹性、来源广泛使用方便、具有很好的生物相容性。此外，最佳的材料在术后不会造成组织的粘连，同时能够促进胸壁修补处的组织重塑。Reconstruction of large-area full-thickness chest wall defects is still a difficult problem for plastic surgery and cardiothoracic surgery. In clinical practice, such operations often require cardiothoracic surgeons to reconstruct and seal the thoracic cavity and reconstruct bony scaffolds. At the same time, soft tissue defects require plastic surgeons to flexibly use appropriate skin flap for graft repair. At present, the factor affecting the reconstruction effect of chest wall defect is still the appropriate selection of materials for chest wall reconstruction. Although there are currently titanium plates, artificial bones, and various meshes to choose from, none of them can fully meet the ideal requirements for chest wall reconstruction. Preclinical research is constantly looking for new materials that are closer to the normal shape and function of the chest wall. With the development of materials science and tissue engineering, a more suitable reconstruction material is bound to significantly improve the postoperative survival of such patients. quality. An ideal chest wall replacement material needs to have excellent tensile strength and elasticity, a wide range of sources, convenient use, and good biocompatibility. In addition, the optimal material does not cause tissue adhesion after surgery, while promoting tissue remodeling at the chest wall repair.
POC是具有极佳弹性的可吸收材料，最初设计用于组织工程小血管的支架材料[JYang，WA R.，AG A.Novel Citric Acid‐Based Biodegradable Elastomers for TissueEngineering.Advanced Materials,2004,16(6):511-516]。POC合成过程中对环境的要求不高，甚至在37℃，就可以合成，这使得POC材料可以和药物或者蛋白结合成为复合体。此外还可以根据需要调节力学强度和分解率，固有表面容易被细胞附着。目前文献报导了内皮祖细胞、心肌细胞、成肌细胞、软骨细胞、平滑肌细胞等细胞的体外共培养，各种细胞和POC材料都有很好的相容性[Hidalgo-Bastida LA,Barry JJ,Everitt NM,er al.Celladhesion and mechanical properties of a flexible scaffold for cardiac tissueengineering.Acta Biomater.2007Jul；3(4):457-62.；MP Prabhakaran，AS Nair，K Dan，et al.Electrospun composite scaffolds containing poly(octanediol-co-citrate)for cardiac tissue engineering&dagger.Biopolymers,2012,97(7):529&ndash；538.；Claire G.Jeong,Huina Zhang,Scott J.Hollister.Three-dimensional poly(1,8-octanediol–co-citrate)scaffold pore shape and permeability effects on sub-cutaneous in vivo chondrogenesis using primary chondrocytes.ActaBiomaterialia,2011；505–514.]。POC is an absorbable material with excellent elasticity, originally designed as a scaffold material for tissue engineering small blood vessels [J Yang, WA R., AG A. Novel Citric Acid-Based Biodegradable Elastomers for Tissue Engineering. Advanced Materials, 2004, 16 (6 ):511-516]. The POC synthesis process does not require high environmental requirements, and it can be synthesized even at 37 °C, which enables the POC material to be combined with drugs or proteins to form a complex. In addition, the mechanical strength and decomposition rate can be adjusted as required, and the inherent surface is easily attached by cells. At present, the in vitro co-culture of endothelial progenitor cells, cardiomyocytes, myoblasts, chondrocytes, smooth muscle cells and other cells has been reported in the literature, and various cells and POC materials have good compatibility [Hidalgo-Bastida LA, Barry JJ, Everitt NM, er al. Celladhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering. Acta Biomater. 2007 Jul;3(4):457-62.; MP Prabhakaran, AS Nair, K Dan, et al. Electrospun composite scaffolds containing poly (octanediol-co-citrate)for cardiac tissue engineering&dagger.Biopolymers,2012,97(7):529–538.;Claire G.Jeong,Huina Zhang,Scott J.Hollister.Three-dimensional poly(1,8-octanediol– co-citrate) scaffold pore shape and permeability effects on sub-cutaneous in vivo chondrogenesis using primary chondrocytes. Acta Biomaterialia, 2011; 505–514.].
静电纺丝技术就是高分子流体静电雾化这样的一种形式，此时雾化分裂出的物质是聚合物微小射流，可以在干燥的环境内运行相当长的距离，最终固化成纳米级别的聚合物纤维。静电纺丝制成的支架有很好的孔隙率，适合细胞的生长。PLA则是目前临床常用的可降解材料。我们通过POC与PLA等比例混合用，用电纺丝技术制备成具有3D结构的生物膜片用于胸壁重建。Electrospinning technology is a form of electrostatic atomization of polymer fluids. At this time, the atomized and split substances are tiny polymer jets, which can run for a long distance in a dry environment and finally solidify into nano-scale polymerization. material fiber. The scaffolds made by electrospinning have good porosity and are suitable for cell growth. PLA is a biodegradable material commonly used in clinical practice. By mixing POC and PLA in equal proportions, we prepared a biofilm with a 3D structure by electrospinning technology for chest wall reconstruction.
脂肪干细胞是成体间充质干细胞的一种，从脂肪之中获取。它具有向三个胚层分化的能力，可在不同的诱导因子作用下分化为多种细胞，同时可以分泌多种细胞因子[Schaffle A，Buchler C.Concise review:adipose tissue-derived stromal cells—bisic and clinical implications for novel cell-based therapies.Stem Cells,2007,25(4)；818-827.]。研究表明脂肪干细胞可以通过多种机制加快创面的愈合，包括旁分泌作用、多向分化能力、促进血管新生等[张元政、邢新、杨超。脂肪来源干细胞修复创面的研究进展。中国美容整形外科杂志。2014,25(12)：742-44.]。ASCs表面仅表达非常少量的主要组织兼容性复合物-Ⅰ(Major histocompability complex-Ⅰ，MHC-Ⅰ)，而主要组织兼容性复合物-Ⅱ(Major histocompability complex-Ⅱ，MHC-Ⅱ)、CD80、CD86和CD40则不表达[Le Blanc,K.and O.Ringden.Immunosuppression by mesenchymal stem cells andclinical experience.J Intern Med,2007.265(5):509-525.]，因此不会激活异体的淋巴免疫反应。ASCs也被证实可以抑制T和B淋巴细胞的活化和增生[Corcione,A.et al.Humanmesenchymal stem cells modulate B-cell function.Blood,2006,107:367-72.]。此外，ASCs还能分泌白介素6(Interleukin-6，IL-6)、集落刺激因子(Colony stimulatingFactor，CSF)等影响树突状细胞的分化、成熟和功能[Djouad,F.et al.mesenchymal stemcells inhibit the differentiation of dendritic cells through an interleukin-6dependent mechanism.Stem Cells,2007,25:2025-32.]。可见ASCs有较低的免疫源性，能减轻炎症反应，异种动物使用也不会引起宿主的免疫反应。因此脂肪干细胞的运用对于加速创伤的愈合，减少炎症反应都将为一系列疾病的治疗提供新的方法。Adipose-derived stem cells are a type of adult mesenchymal stem cells obtained from fat. It has the ability to differentiate into three germ layers, can differentiate into a variety of cells under the action of different inducing factors, and can secrete a variety of cytokines at the same time [Schaffle A, Buchler C. Concise review:adipose tissue-derived stromal cells—bisic and clinical implications for novel cell-based therapies. Stem Cells, 2007, 25(4);818-827.]. Studies have shown that adipose stem cells can accelerate wound healing through various mechanisms, including paracrine action, multi-directional differentiation ability, and promotion of angiogenesis [Zhang Yuanzheng, Xing Xin, Yang Chao. Research progress of adipose-derived stem cells in wound repair. Chinese Journal of Aesthetic Plastic Surgery. 2014, 25(12):742-44.]. ASCs only express a very small amount of major histocompability complex-I (MHC-I) on the surface of ASCs, while major histocompability complex-II (MHC-II), CD80, CD86 and CD40 are not expressed [Le Blanc, K. and O. Ringden. Immunosuppression by mesenchymal stem cells and clinical experience. J Intern Med, 2007. 265 (5): 509-525.], so it will not activate the allogeneic lymphoid immune response. ASCs have also been shown to inhibit the activation and proliferation of T and B lymphocytes [Corcione, A. et al. Humanmesenchymal stem cells modulate B-cell function. Blood, 2006, 107:367-72.]. In addition, ASCs can also secrete interleukin-6 (Interleukin-6, IL-6), colony stimulating factor (Clony stimulating Factor, CSF), etc. to affect the differentiation, maturation and function of dendritic cells [Djouad, F. et al. mesenchymal stem cells inhibit the differentiation of dendritic cells through an interleukin-6 dependent mechanism. Stem Cells, 2007, 25:2025-32.]. It can be seen that ASCs have low immunogenicity and can reduce the inflammatory response, and the use of xenogeneic animals will not cause the immune response of the host. Therefore, the use of adipose stem cells to accelerate wound healing and reduce inflammatory responses will provide new methods for the treatment of a series of diseases.
中国专利文献CN 103877622A公开了一种静电纺丝纳米纤维-细胞外基质复合材料及其制备方法和应用，制备方法包括如下步骤：S1.制备生物降解材料的静电纺丝纤维膜，所述生物降解材料为聚己内酯、左旋聚乳酸、聚乳酸-羟基乙酸共聚物；S2.将步骤S1制得的静电纺丝纤维膜平铺在细胞培养容器中，用培养基浸泡，然后接种干细胞，于CO2培养箱中培养5～10天，每隔2～3天更换培养基，接着进行脱细胞处理，最后干燥即可得到静电纺丝纳米纤维-细胞外基质复合材料。Chinese patent document CN 103877622A discloses an electrospinning nanofiber-extracellular matrix composite material and a preparation method and application thereof. The preparation method includes the following steps: S1. preparing an electrospinning fiber membrane of a biodegradable material, the biodegradable The materials are polycaprolactone, L-polylactic acid, and polylactic acid-glycolic acid copolymer; S2. The electrospinning fiber membrane prepared in step S1 is flattened in a cell culture vessel, soaked in culture medium, and then inoculated with stem cells. The electrospinning nanofiber-extracellular matrix composite material can be obtained by culturing in a CO2 incubator for 5-10 days, changing the medium every 2-3 days, then performing decellularization treatment, and finally drying.
目前尚无文献报道有关ASCs膜片结合POC-PLA补片复合体修复胸壁缺损的研究及应用。At present, there is no literature report on the research and application of ASCs patch combined with POC-PLA patch complex to repair chest wall defects.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种新型的生物补片材料，本发明的另一目的在于提供一种修复组织缺损的方法，以及该生物补片材料和方法在胸壁全层缺损中的应用。The purpose of the present invention is to provide a novel biological patch material, and another purpose of the present invention is to provide a method for repairing tissue defects, and the application of the biological patch material and method in full-thickness chest wall defects.
本发明的第一方面，提供一种ASCs结合POC-PLA静电纺丝补片复合体，由POC-PLA静电纺丝补片和ASCs细胞膜片组成，所述的POC-PLA静电纺丝补片中POC和PLA的质量比为1：1；所述的ASCs细胞膜片是采用细胞膜片技术将ASCs均匀的种植于POC-PLA静电纺丝补片之上。The first aspect of the present invention provides an ASCs combined with a POC-PLA electrospinning patch composite, which is composed of a POC-PLA electrospinning patch and an ASCs cell membrane sheet, wherein the POC-PLA electrospinning patch is The mass ratio of POC and PLA was 1:1; the ASCs cell sheet was uniformly planted on the POC-PLA electrospinning patch by using the cell sheet technology.
本发明的第二方面，提供上述ASCs结合POC-PLA静电纺丝补片复合体的制备方法，包括以下步骤：The second aspect of the present invention provides a preparation method of the above-mentioned ASCs combined with POC-PLA electrospinning patch complex, comprising the following steps:
A)POC-PLA静电纺丝补片的制备：制备POC预聚体，以三氟乙醇为溶剂，按质量比1：1的配比配制POC预聚体与PLA的溶液，其中PLA质量分数为10％，用上述溶液进行静电纺丝，静电纺丝得到的纤维膜进行固化、灭菌，得到POC-PLA静电纺丝补片；A) Preparation of POC-PLA electrospinning patch: To prepare POC prepolymer, use trifluoroethanol as solvent to prepare a solution of POC prepolymer and PLA in a mass ratio of 1:1, where the mass fraction of PLA is 10%, electrospinning with the above solution, and curing and sterilizing the fiber membrane obtained by electrospinning to obtain a POC-PLA electrospinning patch;
B)ASCs膜片结合POC-PLA静电纺丝补片复合体的制备：分离培养ASCs，取第3代ASCs制备附着在纤维蛋白薄片上的细胞膜片，将纤维蛋白薄片含细胞膜片一侧向下放于补片之上，在培养箱内37℃下孵育30分钟；轻轻掀去纤维蛋白薄片，取出膜片复合体；将膜片复合体置于细胞培养箱内孵育24小时；取出膜片复合体，用PBS轻轻冲洗去除培养基，得到所述的静电纺丝补片复合体。B) Preparation of ASCs sheet combined with POC-PLA electrospinning patch complex: ASCs were isolated and cultured, and the third-generation ASCs were taken to prepare cell sheets attached to the fibrin sheet, and the side of the fibrin sheet containing the cell sheet was placed down. On the patch, incubate at 37°C for 30 minutes in the incubator; gently lift off the fibrin sheet, take out the patch complex; place the patch complex in the cell culture incubator for 24 hours; take out the patch complex body, and gently rinsed with PBS to remove the medium to obtain the electrospinning patch complex.
优选的，所述的步骤A中POC预聚体的制备方法包括：按摩尔比1：1将柠檬酸和1,8-辛二醇加入到容器中，在氮气保护下，在油浴中加热至160℃，搅拌10min至粉末全部熔融；然后在140℃、常压下聚合约40min，得到预聚体；趁热迅速将预聚体移出，依次加入乙醇溶解预聚体及蒸馏水沉淀出预聚体，反复清洗数次，以完全除去未反应的单体，得到较为纯净的POC预聚体。Preferably, the preparation method of the POC prepolymer in the step A includes: adding citric acid and 1,8-octanediol into a container in a molar ratio of 1:1, heating in an oil bath under nitrogen protection to 160°C, stir for 10 minutes until the powder is completely melted; then polymerize at 140°C and normal pressure for about 40 minutes to obtain a prepolymer; quickly remove the prepolymer while hot, and sequentially add ethanol to dissolve the prepolymer and distilled water to precipitate the prepolymer. The polymer was washed repeatedly for several times to completely remove the unreacted monomer, and a relatively pure POC prepolymer was obtained.
优选的，所述的步骤B包括以下步骤：Preferably, described step B comprises the following steps:
(1)取分离培养的第3代ASCs，按照传代相同的方法消化细胞后，以1×106接种于温敏培养皿，加入含10％FBS的完全培养基；(1) Take the 3rd generation ASCs isolated and cultured, digest the cells according to the same method of passage, inoculate 1×106 in a temperature-sensitive petri dish, and add complete medium containing 10% FBS;
(2)细胞恒温培养箱5％CO2，37℃条件下培养；(2) Cells are incubated at 37°C in a constant temperature incubator with 5% CO2 ;
(3)待细胞融合长满后，吸走温敏培养皿内培养基，加适量PBS湿润细胞膜片。将配套的纤维蛋白薄片轻轻放置于细胞膜片表面，注意不要产生气泡，温敏培养皿置于25℃孵化10分钟；(3) After the cells are confluent and full, the medium in the temperature-sensitive petri dish is aspirated, and an appropriate amount of PBS is added to wet the cell membrane. Gently place the matching fibrin sheet on the surface of the cell membrane, taking care not to generate air bubbles, and incubate the temperature-sensitive culture dish at 25°C for 10 minutes;
(4)用无菌镊子轻轻掀起纤维蛋白薄片，镜下观察温敏培养皿是否有残余的细胞；(4) Gently lift the fibrin sheet with sterile tweezers, and observe under the microscope whether there are residual cells in the temperature-sensitive culture dish;
(5)POC-PLA补片剪裁4×4cm大小，在PBS中浸泡2小时后，将纤维蛋白薄片含细胞膜片一侧向下放于补片之上，加1ml培养基，在培养箱内37℃下孵育30分钟；(5) Cut the POC-PLA patch to a size of 4 × 4 cm, soak it in PBS for 2 hours, place the side of the fibrin sheet containing the cell membrane down on the patch, add 1 ml of culture medium, and place it in an incubator at 37°C Incubate for 30 minutes;
(6)轻轻掀去纤维蛋白薄片，取出膜片复合体；(6) Gently lift off the fibrin sheet, and take out the membrane sheet complex;
(7)将膜片复合体置于细胞培养箱内孵育24小时；(7) The membrane sheet complex is placed in a cell incubator and incubated for 24 hours;
(8)取出膜片复合体，用PBS轻轻冲洗去除培养基，得到所述的静电纺丝补片复合体。(8) Take out the patch complex, and gently rinse with PBS to remove the medium to obtain the electrospinning patch complex.
本发明的第三方面，提供上述ASCs结合POC-PLA静电纺丝补片复合体在制备胸壁缺损重建材料中的应用。The third aspect of the present invention provides the application of the above-mentioned ASCs combined with the POC-PLA electrospinning patch complex in the preparation of chest wall defect reconstruction materials.
本发明的构思如下：本发明基于ASCs来源广泛，具有向多胚层分化、促进组织修复的能力，与弹性良好的可吸收材料POC-PLA合成生物补片复合体，通过肉眼及镜下的形态学观察，及相关炎症指标和增殖凋亡的检测其生物相容性，并建立动物模型，研究运用此生物补片复合体修复胸壁缺损对机体的影响。The concept of the present invention is as follows: the present invention is based on a wide range of sources of ASCs, has the ability to differentiate into multiple germ layers and promote tissue repair, and synthesize biological patch complexes with the absorbable material POC-PLA with good elasticity. Observation and related inflammatory indicators and detection of proliferation and apoptosis of its biocompatibility, and establishment of animal models to study the effect of using this biological patch complex to repair chest wall defects on the body.
本发明的主要技术方案包括三个部分：The main technical scheme of the present invention includes three parts:
第一部分，rASCs分离与鉴定。我们取兔颈背部的脂肪，采用酶消化法分离出rASCs。在分离的过程中，需要注意的是对消化前期脂肪的预处理，在获取脂肪之后，对脂肪组织充分的冲洗和修剪可以有效提高获取的ASCs纯度。用流式细胞术对上述方法分离成功的ASCs进行细胞表型鉴定，结果表明CD29、CD44、CD90的阳性表达率分别为92％、38％、93.5％(见图1)。证明用酶消化法能非常高效的获得较高纯度的ASCs。The first part, isolation and identification of rASCs. We took the fat from the back of the rabbit's neck and isolated rASCs by enzymatic digestion. In the process of isolation, attention should be paid to the pretreatment of the pre-digested fat. After the fat is obtained, adequate washing and trimming of the adipose tissue can effectively improve the purity of the obtained ASCs. Flow cytometry was used to identify the cell phenotype of the successfully isolated ASCs. The results showed that the positive expression rates of CD29, CD44, and CD90 were 92%, 38%, and 93.5%, respectively (see Figure 1). It is proved that the enzymatic digestion method can obtain high-purity ASCs very efficiently.
实验中我们发现ASCs的细胞增值能力会随着传代数的增多而渐渐下降，因此在使用rASCs时往往使用代数较低的细胞。我们取第3代rASCs进行了多向分化的检测。可以看到在诱导培养基的作用下，rASCs向软骨、骨和脂肪都有较高的转化率。前三代MTT检测和细胞生长曲线都表明，第3代rASCs细胞活性佳，有很好的干性(见图2)。后续的实验中，我们将取第3代ASCs作为种子细胞进行实验。In the experiment, we found that the cell proliferation ability of ASCs will gradually decrease with the increase of passage number, so cells with lower passage number are often used when using rASCs. We took the 3rd generation rASCs to detect multi-directional differentiation. It can be seen that under the action of the induction medium, rASCs have higher conversion rates to cartilage, bone and fat. The MTT assays and cell growth curves of the first three passages showed that the third passage rASCs had good cell viability and good stemness (see Figure 2). In the follow-up experiments, we will use the third generation ASCs as seed cells for experiments.
第二部分，rASCs膜片结合POC-PLA静电纺丝补片复合体的建立与材料安全性检测。将POC与PLA等比例混合后，静电纺丝形成具有一定力学性能的可降解膜片作为全层胸壁重建的材料(见图3)。在膜片靠近胸腔侧，种植一层脂肪干细胞膜片，防止补片与肺脏之间的粘连。The second part, the establishment of rASCs membrane combined with POC-PLA electrospinning patch complex and the material safety testing. After mixing POC and PLA in equal proportions, a degradable membrane with certain mechanical properties was formed by electrospinning as a material for full-thickness chest wall reconstruction (see Figure 3). On the side of the patch close to the thoracic cavity, a layer of adipose stem cell membrane was planted to prevent adhesion between the patch and the lungs.
在补片的力学测试中，相比较目前临床常用的e-PTFE补片，厚度较厚的POC-PLA静电纺丝补片与薄层的e-PTFE补片表现出相似的弹力系数。而通过抗张强度和杨氏模量的比较，可以发现POC-PLA材料较e-PTFE有更好的弹力(见图4)。而在使用静电纺丝技术同样赋予补片很好的三维结构，在电镜扫描的结果中，我们可以看到补片的孔隙直径约在纳米级别。这样的三维孔隙结构使得ASCs能很好的粘附于材料表面。在ASCs细胞种植于材料的24小时内，就能观察到细胞粘附于材料表面。在移植入体内后，我们可见看到补片在体内被组织所包裹，材料内部孔隙可见细胞长入。植入第一周的补片CD31染色就可以看到周边有大量新生的血管，在第四周的组织化学染色我们可以看见更多的血管生成以及长入(见图5)。这些特性能够证明补片能很好的作为支架，参与到组织的重建。In the mechanical test of the patch, compared with the currently commonly used e-PTFE patch, the thicker POC-PLA electrospinning patch and the thin e-PTFE patch showed similar elastic coefficients. By comparing the tensile strength and Young's modulus, it can be found that the POC-PLA material has better elasticity than e-PTFE (see Figure 4). The electrospinning technology also gives the patch a good three-dimensional structure. In the results of electron microscope scanning, we can see that the pore diameter of the patch is about nanometers. Such a three-dimensional pore structure enables ASCs to adhere well to the material surface. Within 24 hours after ASCs cells were seeded on the material, cell adhesion to the material surface was observed. After being transplanted into the body, we can see that the patch is wrapped by tissue in the body, and cells grow into the pores inside the material. The patch CD31 staining in the first week of implantation could see a large number of new blood vessels around it, and in the fourth week of histochemical staining we could see more angiogenesis and ingrowth (see Figure 5). These properties can prove that the mesh can be used as a scaffold to participate in tissue reconstruction.
在组织相容性的测试中，我们可以发现，组织移植入的早期存在轻度的异物反应，膜片表面可以看到巨噬细胞的浸润，IL-6、TGFβ与TNFα等指标都显示升高。在早期膜片附近的细胞表现出增强的增殖信号水平，对caspase-3组织化学染色我们可以发现，膜片周边细胞凋亡水平较正常增强，这可能是因为早期的炎症反应所致，TUNEL染色可以看到少量的凋亡细胞。而一个月左右的样本我们可以发现炎症反应已经很轻，增殖与凋亡也接近于正常水平。(见图6、7)In the histocompatibility test, we can find that there is a mild foreign body reaction in the early stage of tissue transplantation, the infiltration of macrophages can be seen on the surface of the membrane, and the indicators such as IL-6, TGFβ and TNFα all show increased . Cells near the membrane at the early stage showed an enhanced level of proliferative signals. On the histochemical staining of caspase-3, we could find that the apoptosis level of the cells around the membrane was enhanced compared with normal, which may be caused by the early inflammatory response. TUNEL staining A small number of apoptotic cells can be seen. In the sample of about a month, we can find that the inflammatory response is very mild, and the proliferation and apoptosis are also close to normal levels. (See Figures 6 and 7)
我们比较了使用细胞膜片技术和直接种植细胞于材料表面，结果证明细胞膜片技术能在很短的时间内将大量的ASCs种植于材料之上，形成膜片样结构覆盖材料表面。共培养方式种植ASCs制备时间更长，而且细胞容易形成局部生长的，相比较而言，膜片技术培养的细胞生长更为均匀。(见图8)We compared the use of cell sheet technology and the direct seeding of cells on the surface of the material, and the results proved that the cell sheet technique can seed a large number of ASCs on the material in a very short time, forming a sheet-like structure covering the surface of the material. The preparation time of ASCs grown in the co-culture method is longer, and the cells are easy to form local growth. In comparison, the growth of cells cultured by the patch technology is more uniform. (see Figure 8)
综上所述，POC-PLA静电纺丝补片有着极佳力学弹性的可吸收补片。细胞膜片技术则将大量的脂肪干细胞均匀的种植于补片之上。这样形成的补片复合体既有很好的力学弹性，又可以促进机体的重建，减轻炎症反应带来的粘连。下面我们将使用这种补片复合体来重建兔的胸壁全层缺损。In conclusion, the POC-PLA electrospun patch is an absorbable patch with excellent mechanical elasticity. The cell patch technology evenly planted a large number of adipose stem cells on the patch. The patch complex thus formed not only has good mechanical elasticity, but also can promote the reconstruction of the body and reduce the adhesion caused by the inflammatory response. Below we will use this patch complex to reconstruct a full-thickness defect of the chest wall in rabbits.
第三部分，rASCs膜片结合POC-PLA电纺丝补片复合体修复兔全层胸壁缺损的研究。我们的动物模型选用新西兰兔。新西兰兔体型相对狭长，胸壁保护胸腔内器官与部分腹腔器官。新西兰兔胸廓面积约5×10cm，参照文献制备模型，缺损部位位于右侧胸壁，面积约3×3cm，按正常成人的胸壁面积换算，约为10×10cm缺损面积，需要使用补片来加固胸壁。(见图9)The third part, the study of rASCs patch combined with POC-PLA electrospinning patch complex to repair full-thickness chest wall defect in rabbits. Our animal model uses New Zealand rabbits. New Zealand rabbits are relatively long and narrow, and the chest wall protects the thoracic organs and part of the abdominal organs. The thoracic area of a New Zealand rabbit is about 5 × 10 cm. The model is prepared with reference to the literature. The defect is located on the right chest wall, with an area of about 3 × 3 cm. Converted to the area of the normal adult chest wall, the defect area is about 10 × 10 cm. Patches are needed to reinforce the chest wall. . (see Figure 9)
我们可以观察到，在一个月的样本中，补片已经被自身组织所包裹，在两个月开始补片慢慢开始裂解，在六个月的样本中，可见补片已经完全吸收完毕，被自身组织所取代(见图10)。组织的力学测试可以看出在8周后弹力系数已经接近正常的胸壁肋间肌的弹性。We can observe that in the one-month sample, the patch has been wrapped by its own tissue, and the patch slowly begins to disintegrate at two months. In the six-month sample, it can be seen that the patch has been completely absorbed and is replaced by its own tissue (see Figure 10). The mechanical test of the tissue shows that the elasticity coefficient of the intercostal muscles of the chest wall is close to that of the normal chest wall after 8 weeks.
成长期的胸壁重建，容易出现胸壁的畸形。在单纯进行肌皮瓣重建的兔子，在8周以上的大体样本上很容易发现胸壁的畸形，包括胸壁塌陷和胸廓移位，这可能跟肌皮瓣萎缩有关。而ePTFE重建和POC-PLA重建在早期均为出现胸廓的异常，24周以后少量的ePTFE重建胸壁出现了轻微的胸骨移位。在早期创面边缘的组织增生减少了创面的面积，但是随着体型增长与胸廓生长速度超过组织增生的量以后，ePTFE又会拉扯胸廓引起胸骨的移位。在POC-PLA材料重建的样本中，均未出现胸壁的畸形和移位。这可能与较轻的炎症反应和自体细胞的重建有关。在连续的观察中可以看到早期炎症细胞的浸润与成纤维细胞的长入，到后期材料边缘有软骨细胞的长入，而表面除了成纤维细胞之外，还可以看到肌纤维样的组织参与了重建。(见图11、12)Reconstruction of the chest wall during the growth period is prone to deformity of the chest wall. In rabbits undergoing musculocutaneous flap reconstruction alone, chest wall deformities, including chest wall collapse and thoracic displacement, were easily found in gross samples older than 8 weeks, which may be related to musculocutaneous flap atrophy. However, both ePTFE reconstruction and POC-PLA reconstruction showed abnormalities of the thorax in the early stage, and a small amount of ePTFE reconstruction showed slight sternal displacement after 24 weeks. In the early stage, tissue proliferation at the edge of the wound reduces the area of the wound, but as the body size increases and the growth rate of the thorax exceeds the amount of tissue proliferation, ePTFE will pull the thorax and cause displacement of the sternum. In the samples reconstructed with POC-PLA material, there was no deformity and displacement of the chest wall. This may be related to a milder inflammatory response and reconstitution of autologous cells. In the continuous observation, the infiltration of inflammatory cells and the ingrowth of fibroblasts can be seen in the early stage, and the ingrowth of chondrocytes can be seen at the edge of the material in the later stage. In addition to fibroblasts, muscle fiber-like tissue can also be seen on the surface. rebuilt. (See Figures 11 and 12)
在术后效果的评估中，我们把肺功能也引入作为一项重要的评估指标。尽管所有动物均没有出现呼吸衰竭、反式呼吸等并发症，但是进行肺功能的检测还是能够发现差异。单纯进行肌皮瓣重建的兔子肺功能在术后多个时间点的检查中，都表现的最差，FVC、FEF25～75％、0.4秒和0.6秒同期率都显著低于其他组。ePTFE作为临床常用的补片，在术后肺功能的恢复有较好的作用，各项指标都接近正常。单纯的POC-PLA补片术后肺功能的恢复介于单纯皮瓣修复和使用ePTFE补片之间。尽管组织学检测POC-PLA补片修复胸壁进行了较好的重建，但是修补区域较为严重的粘连，可能是导致肺功能减少的原因。结合干细胞的POC-PLA补片复合体在所有组别中肺功能恢复的表现最好。在术后第24周的肺功能检测中，已经接近于正常水平。(见图13)In the evaluation of postoperative effect, we also introduced lung function as an important evaluation index. Although none of the animals developed respiratory failure, trans-breathing and other complications, differences in lung function tests could still be found. The pulmonary function of rabbits who underwent musculocutaneous flap reconstruction alone showed the worst performance at multiple time points after operation, and the FVC, FEF25-75%, 0.4 seconds and 0.6 seconds were significantly lower than other groups. As a commonly used patch in clinic, ePTFE has a good effect on the recovery of postoperative pulmonary function, and all indicators are close to normal. The recovery of lung function after simple POC-PLA mesh was between that of simple flap repair and ePTFE mesh. Although the POC-PLA patch repaired the chest wall with histological examination and carried out good reconstruction, the more serious adhesions in the repaired area may be the reason for the reduction of lung function. The POC-PLA patch complex combined with stem cells performed best in the recovery of lung function of all groups. In the 24-week postoperative pulmonary function test, it was close to the normal level. (see Figure 13)
POC-PLA在体内的降解率较体外更快。在术后4周的样本中，大体可见补片已经局部出现了裂缝；12周的样本中，大部分补片已经出现龟裂；术后24周的样本中，补片已经被分解为小块的碎片。CD31免疫组织化学染色可见补片周边有密集的血管新生，在术后12周可以看见有血管长入补片裂痕间隙之中。复合了干细胞膜片的样本可见到早期的血管密度更高，这可能加速了胸壁重建的速度(见图14)。The degradation rate of POC-PLA was faster in vivo than in vitro. In the 4-week postoperative sample, it is generally seen that the patch has been partially cracked; in the 12-week sample, most of the patch has been cracked; in the 24-week postoperative sample, the patch has been broken down into small pieces shards. CD31 immunohistochemical staining showed dense angiogenesis around the patch, and 12 weeks after surgery, blood vessels could be seen growing into the gap of the patch. Higher blood vessel density was seen early in the composite stem cell patch samples, which may have accelerated the rate of chest wall reconstruction (see Figure 14).
ePTFE补片有较好的防粘连效果，粘连评分接近单纯肌皮瓣修复，仅仅在少数样本里发现比较严重的粘连。POC-PLA补片比较容易产生胸膜粘连，在所有的样本中，均存在着不同程度的粘连情况。这种粘连的产生，可能与早期的炎症反应有关。Western Blot以及免疫组化均表现出TGFβ、IL6、TNFα等炎症指标的升高。而在应用干细胞膜片之后，胸膜粘连的状况明显改善，炎症指标也相应的下调。此外，POC-PLA较好的细胞附着性[Hidalgo-Bastida LA,Barry JJ,Everitt NM,et al.Cell adhesion and mechanical propertiesof a flexible scaffold for cardiac tissue engineering.Acta Biomater.2007 Jul；3(4):457-62.]，也可能是造成胸膜粘连的原因之一。干细胞膜片有效的阻止了脏层胸膜与材料早期的直接接触，减少材料对胸腔内气管的吸附。同时干细胞分泌的细胞因子，也可能加速了内皮样细胞和纤维细胞对材料的包裹，减少后期粘连的产生。(图15、16)The ePTFE mesh has a good anti-adhesion effect, and the adhesion score is close to that of simple musculocutaneous flap repair. Only a few samples were found to have serious adhesions. POC-PLA patches are more prone to pleural adhesions, and there are different degrees of adhesion in all samples. The generation of this adhesion may be related to the early inflammatory response. Western Blot and immunohistochemistry showed the increase of TGFβ, IL6, TNFα and other inflammatory indicators. After the application of the stem cell patch, the pleural adhesions were significantly improved, and the inflammatory indexes were also down-regulated accordingly. In addition, POC-PLA has better cell adhesion [Hidalgo-Bastida LA, Barry JJ, Everitt NM, et al. Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering. Acta Biomater. 2007 Jul; 3(4): 457-62.], may also be one of the causes of pleural adhesions. The stem cell sheet effectively prevents the early direct contact between the visceral pleura and the material, and reduces the adsorption of the material to the trachea in the thoracic cavity. At the same time, the cytokines secreted by stem cells may also accelerate the encapsulation of the material by endothelial-like cells and fibroblasts and reduce the generation of late adhesion. (Figure 15, 16)
本发明优点在于：The advantages of the present invention are:
本发明采用了弹性体POC材料为主体，加入等比例的PLA，通过静电纺丝技术共纺出具有3D纳米孔隙结构的补片，与ASCs细胞膜片复合构建成补片复合体，POC-PLA制成的补片有良好的生物相容性和可控的力学效应，细胞膜片技术则将大量的脂肪干细胞均匀的种植于补片之上，这样形成的补片复合体既有很好的力学弹性，又可以促进机体的重建，减轻炎症反应带来的粘连，促进胸壁的重构过程。The invention adopts the elastomer POC material as the main body, adds equal proportions of PLA, and co-spins a patch with a 3D nano-pore structure through the electrospinning technology, which is compounded with the ASCs cell membrane to form a patch complex, which is made of POC-PLA. The obtained patch has good biocompatibility and controllable mechanical effect, and the cell membrane technology evenly planted a large number of adipose stem cells on the patch, so that the formed patch complex has good mechanical elasticity. , and can promote the reconstruction of the body, reduce the adhesion caused by the inflammatory response, and promote the reconstruction process of the chest wall.
附图说明Description of drawings
图1.rASCs细胞的表型鉴定。Figure 1. Phenotypic identification of rASCs cells.
图2.rASCs MTT检测。Figure 2. rASCs MTT assay.
图3.(A)POC-PLA电纺丝补片大体形态。(B)POC-PLA电纺丝补片电镜下形态，放大倍数2000倍。Figure 3. (A) General morphology of POC-PLA electrospun patch. (B) The morphology of POC-PLA electrospun patch under electron microscope, magnification 2000 times.
图4.(A)POC-PLA电纺丝补片降解率(B)POC-PLA电纺丝补片拉伸系数检测。Figure 4. (A) POC-PLA electrospun patch degradation rate (B) POC-PLA electrospun patch tensile coefficient detection.
图5.H&E组织染色(200×)。Figure 5. H&E tissue staining (200x).
图6.SD大鼠免疫组化，从上到下分别为CD68、IL-6、TNFα和TGF-β(200×)。Figure 6. SD rat immunohistochemistry, from top to bottom, CD68, IL-6, TNFα and TGF-β (200×).
图7.SD大鼠免疫组化，从上到下分别为Ki67、PCNA、caspase3和TUNEL(200×)。Figure 7. SD rat immunohistochemistry, Ki67, PCNA, caspase3 and TUNEL (200×) from top to bottom.
图8.上排图为细胞与材料种植后混合培养5天后电镜照片，放大倍数从左到右分别为2000 1000 500；下排图为ASCs膜片复合补片细胞培养箱内静置2h后电镜照片，放大倍数从左到右分别为2000×1000×500×。Figure 8. The top row shows the electron microscope photos after 5 days of mixed culture of cells and materials after planting, the magnifications are 2000 1000 500 from left to right; the bottom row is the electron microscope after the ASCs membrane composite patch cell incubator has been standing for 2 hours Photos, magnifications are 2000×1000×500× from left to right.
图9.图全层胸壁缺损模型建立与重建。(A)设计手术切口(B)切开皮肤剥离胸大肌至胸壁(C)切除3×3cm大小胸壁，含2-3根肋骨(D)ePTFE补片封闭胸壁缺口(E)POC-PLA补片封闭胸部缺口(F)胸大肌皮瓣覆盖补片，缝合创面。Figure 9. Figure 9. Model establishment and reconstruction of full-thickness chest wall defect. (A) Design of the surgical incision (B) Cut the skin and peel the pectoralis major muscle to the chest wall (C) Excise the chest wall with a size of 3 × 3 cm, including 2-3 ribs (D) The ePTFE patch closes the chest wall gap (E) POC-PLA patch The pectoralis major myocutaneous flap was used to close the chest gap (F), and the patch was covered, and the wound was sutured.
图10.兔组织样本H&E染色。Figure 10. H&E staining of rabbit tissue samples.
图11.兔组织样本masson染色。Figure 11. Masson staining of rabbit tissue samples.
图12.兔组织样本EVG染色。Figure 12. EVG staining of rabbit tissue samples.
图13.术后呼吸功能指标，左图为术后FVC，有图为术后FEV0.4/FVC％。Figure 13. Postoperative respiratory function indicators, the left picture shows the postoperative FVC, and the picture on the left shows the postoperative FEV0.4/FVC%.
图14.兔组织样本CD31免疫组化染色。Figure 14. CD31 immunohistochemical staining of rabbit tissue samples.
图15.肺粘连解剖所见。Figure 15. Anatomical findings of lung adhesions.
图16.胸膜粘连指数。Figure 16. Pleural adhesion index.
图17.POC预聚体反应式，R1、R2、R3和R4为柠檬酸和1,8-辛二醇。Figure 17. POC prepolymer reaction formula, R1, R2, R3 and R4 are citric acid and 1,8-octanediol.
具体实施方式Detailed ways
下面结合实施例对本发明提供的具体实施方式作详细说明。The specific embodiments provided by the present invention will be described in detail below with reference to the examples.
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法，或按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods. In the following examples, the experimental methods without specific conditions are either in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
实施例1：rASCs的分离与鉴定Example 1: Isolation and identification of rASCs
1、脂肪组织的处理：无菌获取的脂肪组织用PBS冲洗两次，洗去表面残留的脂肪组织、血凝块、红细胞等。然后把脂肪组织呈放在10cm的培养皿中，加入20ml PBS溶液，在室温的条件下处理5-10min，即用眼科剪刀和医用镊子仔细除去残余的真皮、结缔组织、血管等。再次清洗脂肪组织，然后移到一个新的培养皿，用整形剪刀剪成体积1mm3不规则的组织块。1. Treatment of adipose tissue: Aseptically obtained adipose tissue was washed twice with PBS to remove residual adipose tissue, blood clots, red blood cells, etc. on the surface. Then put the adipose tissue in a 10cm petri dish, add 20ml of PBS solution, and treat at room temperature for 5-10min, that is, carefully remove the residual dermis, connective tissue, blood vessels, etc. with ophthalmic scissors and medical forceps. The adipose tissue was washed again, then moved to a new petri dish and cut into irregular tissue pieces of1 mm volume with plastic scissors.
2、I型胶原酶配置：电子天平称取100mgI型胶原酶，溶于100mlDMEM培养基，用0.22um滤器除菌。2. Configuration of type I collagenase: Weigh 100 mg of type I collagenase on an electronic balance, dissolve it in 100 ml of DMEM medium, and sterilize it with a 0.22um filter.
3、脂肪组织的酶解：在剪碎成浆糊状的脂肪组织中加入等体积0.1％I型胶原酶，37℃恒温水浴振荡消化50min。3. Enzymatic hydrolysis of adipose tissue: add an equal volume of 0.1% type I collagenase to the adipose tissue cut into paste, and digest with shaking in a constant temperature water bath at 37°C for 50 minutes.
4、离心：消化完全后，加入等量体积的完全培养基(低糖DMEM+10％胎牛血清+1％双抗)中和，500g高速离心10min，取离心后的细胞沉淀层。4. Centrifugation: After digestion is complete, add an equal volume of complete medium (low glucose DMEM + 10% fetal bovine serum + 1% double antibody) to neutralize, centrifuge at 500g for 10 minutes at high speed, and take the cell pellet after centrifugation.
5、加入PBS吹打成细胞悬液后，放入离心机1000r/min低速离心，弃上清。此步骤需重复洗涤两次。5. After adding PBS, pipetting to form a cell suspension, centrifuge at low speed at 1000r/min in a centrifuge, and discard the supernatant. This step needs to be repeated twice.
6、过滤：加入PBS吹打成细胞悬液后，用70目滤网过滤结缔组织至新离心管，离心后加入完全培养基，制成细胞悬液。6. Filtration: After adding PBS, pipetted into a cell suspension, filter the connective tissue with a 70-mesh filter to a new centrifuge tube, and add complete medium after centrifugation to prepare a cell suspension.
7、细胞悬液用细胞计数板计数，按1.0×105/ml密度接种在培养皿内，标准环境下培养，5％CO2，37℃。7. Count the cell suspension with a cell counting plate, inoculate it in a petri dish at a density of 1.0×105 /ml, and cultivate in a standard environment, 5% CO2 , at 37°C.
8、24小时后半量更换培养基，弃去未贴壁的细胞。8. After 24 hours, half of the medium was replaced, and the unadherent cells were discarded.
9、rASCs的培养：当看见培养基颜色发生变化需要进行换液，细胞移液管吸去变色的陈旧培养基，用PBS轻轻冲洗培养皿后加入适量新培养基。9. Cultivation of rASCs: When the color of the medium changes, the medium needs to be changed. The cell pipette removes the discolored old medium, gently rinses the petri dish with PBS, and then adds an appropriate amount of new medium.
10、rASCs的传代：细胞长至密度80％进行传代，用PBS轻轻冲洗培养皿洗去失活细胞，加入0.25％胰酶消化至细胞半贴壁状态后，加入全培养基终止消化后，轻轻吹打下细胞。移送至离心机以1000r/min低速离心，弃上清。加入PBS吹打成细胞悬液后，放入离心机1000r/min低速离心，弃上清。加入适量全培养基吹打成细胞悬液后移植新培养皿。10. Passaging of rASCs: the cells were passaged when the density reached 80%, and the culture dish was gently washed with PBS to remove the inactivated cells. After the cells were digested with 0.25% trypsin until the cells were semi-adherent, the digestion was terminated by adding complete medium. Gently pipet down the cells. Transfer to a centrifuge at a low speed of 1000 r/min, and discard the supernatant. After adding PBS, pipetting to form a cell suspension, centrifuging at low speed at 1000 r/min in a centrifuge, and discarding the supernatant. Add an appropriate amount of complete medium and pipette into a cell suspension and then transplant into a new culture dish.
本实验中，分离的rASCs细胞使用镜下形态学观察、流式细胞技术和多向诱导分化来鉴定rASCs纯度。In this experiment, the isolated rASCs cells were identified by microscopic morphological observation, flow cytometry and multi-directional induced differentiation to identify the purity of rASCs.
镜下形态学观察，取第三代rASCs置于倒置显微镜下观察细胞外形，注意细胞的形态、大小、细胞核状态等。For morphological observation under microscope, the third-generation rASCs were placed under an inverted microscope to observe the shape of the cells, paying attention to the shape, size, and nuclear state of the cells.
实施例2：rASCs膜片结合POC-PLA电纺丝补片复合体的建立Example 2: Establishment of rASCs patch combined with POC-PLA electrospinning patch complex
(一)POC-PLA静电纺丝补片制备(1) Preparation of POC-PLA electrospinning patch
POC-PLA静电纺丝补片制备由东华大学材料学院吉亚丽教授、朱蕾完成，制备方法如下：The preparation of POC-PLA electrospinning patch was completed by Professor Ji Yali and Zhu Lei from the School of Materials, Donghua University. The preparation method is as follows:
1、预聚体的制备：1. Preparation of prepolymer:
按摩尔比1：1将柠檬酸和1,8-辛二醇加入到三口圆底烧瓶中，在氮气保护下，在油浴中加热至160℃，电动搅拌10min至粉末全部熔融。然后在140℃、常压下聚合约40min，得到具有一定分子量的预聚体。趁热迅速将预聚体移出三口烧瓶，转移至大烧杯中，依次加入乙醇(溶解预聚体)及蒸馏水(沉淀出预聚体)，反复清洗数次，以完全除去未反应的单体，可得到较为纯净的POC预聚体。反应式见图17。Add citric acid and 1,8-octanediol into a three-necked round-bottomed flask at a molar ratio of 1:1, heat to 160 °C in an oil bath under nitrogen protection, and stir electrically for 10 min until the powder is completely melted. Then, the polymer was polymerized at 140° C. and normal pressure for about 40 minutes to obtain a prepolymer with a certain molecular weight. Quickly remove the prepolymer from the three-necked flask while it is still hot, transfer it to a large beaker, add ethanol (dissolving the prepolymer) and distilled water (precipitating the prepolymer) in turn, and wash it several times to completely remove unreacted monomers. A relatively pure POC prepolymer can be obtained. The reaction formula is shown in Figure 17.
2、预聚体的静电纺丝：2. Electrospinning of prepolymer:
以三氟乙醇为溶剂，按质量比1：1的配比配制POC预聚体与PLA的溶液，其中PLA质量分数为10％，用上述溶液进行静电纺丝。纺丝之前，需用磁力搅拌器对溶液进行24小时的充分搅拌，以确保溶解完全。将配制完成的溶液装入5mL塑料注射器中，静置以去除注射器中的气泡，然后将注射器置于推进器之上，针尖部分连接静电发生器的正极，接收装置上包裹铝箔纸，固定轴承处接地，调节针尖与接收器之间的距离。开启推进器电源，设置流速，待针尖内溶液稳定均匀流出后，开启静电发生器电源，调节到合适电压，开始纺丝。纺丝结束后，关闭电源。将静电纺丝所得到的覆盖有纤维膜的铝箔取下，置于真空干燥箱中于一定温度下固化。Using trifluoroethanol as a solvent, a solution of POC prepolymer and PLA was prepared in a mass ratio of 1:1, wherein the mass fraction of PLA was 10%, and the above solution was used for electrospinning. Before spinning, the solution needs to be thoroughly stirred with a magnetic stirrer for 24 hours to ensure complete dissolution. Put the prepared solution into a 5mL plastic syringe, let it stand to remove air bubbles in the syringe, then place the syringe on the pusher, connect the needle tip to the positive electrode of the electrostatic generator, wrap the receiving device with aluminum foil, and fix the bearing. Ground, adjust the distance between the tip of the needle and the receiver. Turn on the power of the propeller, set the flow rate, and after the solution in the needle tip flows out stably and evenly, turn on the power of the electrostatic generator, adjust the voltage to an appropriate voltage, and start spinning. After spinning is finished, turn off the power. The aluminum foil covered with the fiber film obtained by electrospinning was removed, and was placed in a vacuum drying oven to cure at a certain temperature.
3、膜片的灭菌：3. Sterilization of the diaphragm:
膜片采用环氧乙烷消毒法灭菌，将膜片剪成合适形状，与环氧乙烷指示条一同装入密封袋，送入仪器灭菌，指示条变色表示灭菌完成。The diaphragm is sterilized by ethylene oxide sterilization. Cut the diaphragm into a suitable shape, put it into a sealed bag together with the ethylene oxide indicator strip, and send it to the instrument for sterilization. The indicator strip changes color to indicate that the sterilization is completed.
(二)rASCs膜片结合POC-PLA静电纺丝补片复合体的制备(2) Preparation of rASCs membrane sheet combined with POC-PLA electrospinning patch complex
1、rASCs的分离和培养：1. Isolation and culture of rASCs:
具体过程见第一部分相应内容。For the specific process, please refer to the corresponding content in the first part.
2、细胞膜片的制备：2. Preparation of cell membrane sheets:
(1)取第3代rASCs，按照传代相同的方法消化细胞后，以1×106接种于温敏培养皿，加入含10％FBS的完全培养基。(1) Take the 3rd generation rASCs, digest the cells according to the same method of passage, inoculate 1×106 in a temperature-sensitive petri dish, and add complete medium containing 10% FBS.
(2)细胞恒温培养箱5％CO2，37℃条件下培养。(2) Cells were cultured in a constant temperature incubator with 5% CO2 at 37°C.
(3)待细胞融合长满后，吸走温敏培养皿内培养基，加适量PBS湿润细胞膜片。将配套的纤维蛋白薄片轻轻放置于细胞膜片表面，注意不要产生气泡，温敏培养皿置于25℃孵化10分钟。(3) After the cells are confluent and full, the medium in the temperature-sensitive petri dish is aspirated, and an appropriate amount of PBS is added to wet the cell membrane. Gently place the matching fibrin sheet on the surface of the cell membrane, taking care not to generate air bubbles, and incubate the temperature-sensitive petri dish at 25°C for 10 minutes.
(4)用无菌镊子轻轻掀起纤维蛋白薄片，镜下观察温敏培养皿是否有残余的细胞。(4) Gently lift the fibrin sheet with sterile tweezers, and observe whether there are residual cells in the temperature-sensitive culture dish under a microscope.
(5)POC-PLA补片剪裁4×4cm大小，在PBS中浸泡2小时后，将纤维蛋白薄片含细胞膜片一侧向下放于补片之上，加1ml培养基，在培养箱内37℃下孵育30分钟。(5) Cut the POC-PLA patch to a size of 4 × 4 cm, soak it in PBS for 2 hours, place the side of the fibrin sheet containing the cell membrane down on the patch, add 1 ml of culture medium, and place it in an incubator at 37°C Incubate for 30 min.
(6)轻轻掀去纤维蛋白薄片，取出膜片复合体。(6) Gently lift off the fibrin sheet, and take out the membrane sheet complex.
(7)将膜片复合体置于细胞培养箱内孵育24小时。(7) Incubate the membrane sheet complex in a cell incubator for 24 hours.
(8)取出膜片复合体，用PBS轻轻冲洗去除培养基。(8) Take out the patch complex, and gently rinse with PBS to remove the medium.
实施例3：rASCs膜片结合POC-PLA电纺丝补片复合体修复兔全层胸壁缺损Example 3: rASCs patch combined with POC-PLA electrospinning patch complex to repair full-thickness chest wall defect in rabbits
选取健康新西兰兔雌性，兔龄为4～5月，体重为2.5-3kg。构建动物模型。Select healthy New Zealand rabbit females, the rabbit age is 4-5 months, and the weight is 2.5-3kg. Build animal models.
1、麻醉：完成抓取固定后，用电子秤称取兔体重，把配置好的1％戊巴比妥钠溶液按0.3ml/Kg的用量耳缘静脉注射麻醉兔。1. Anesthesia: After grasping and fixing, the rabbit was weighed with an electronic scale, and the prepared 1% sodium pentobarbital solution was injected into the ear vein at a dosage of 0.3 ml/Kg to anesthetize the rabbit.
2、固定：将麻醉成功的兔仰卧固定于兔解剖板，四肢固定于固定器，兔充分舒展，暴露整个胸部。2. Fixation: Fix the successfully anesthetized rabbit supine on the rabbit dissection board, fix the limbs on the fixator, and fully stretch the rabbit to expose the entire chest.
3、备皮：用动物剃毛推剔除胸部的毛，上至下颚，下至肋弓，两侧至侧胸壁近上肢关节处，右侧胸壁手术区域可适量剔除右侧上肢毛，充分暴露手术区。3. Skin preparation: Use animal shaving to remove the hair on the chest, up to the lower jaw, down to the costal arch, on both sides to the side of the chest wall near the joints of the upper extremity, and the right upper extremity can be removed from the surgical area of the right chest wall to fully expose the surgery. Area.
4、消毒：兔脱毛区域和其下方相应的解剖台区域用碘伏常规消毒3遍，最后用75％酒精消毒1遍脱色。4. Disinfection: The rabbit depilation area and the corresponding dissection table area below it were routinely disinfected with iodophor 3 times, and finally sterilized with 75% alcohol once for decolorization.
5、标记铺单：在右侧胸壁第5肋骨与胸骨交界处为起点，沿肋骨做一长约6cm切口线，常规铺单(图9)。5. Marking and laying: starting from the junction of the fifth rib on the right side of the chest wall and the sternum, make an incision line about 6 cm long along the rib, and routinely laying the sheet (Figure 9).
6、气管插管：将兔头部向上抬起，保持气管呈水平状态。使用3.0婴儿气管导管盲视下经兔口腔插入气管，插入深度为12.5±1.5cm。插入到位后，使用注射器向气囊注入1ml空气，观察气囊鼓起。气管导管接小动物呼吸机，维持潮气量12ml/kg，频率50次/min。6. Tracheal intubation: lift the rabbit's head up to keep the trachea in a horizontal state. A 3.0 infant tracheal tube was inserted into the trachea through the rabbit's mouth under blind vision, and the insertion depth was 12.5±1.5cm. Once inserted in place, use a syringe to inject 1ml of air into the balloon and watch the balloon bulge. The tracheal tube was connected to a small animal ventilator to maintain a tidal volume of 12ml/kg and a frequency of 50 times/min.
7、全层胸壁缺损模型制作：沿切口线切开皮肤，暴露胸大肌，顺着胸大肌肌肉走向切开胸大肌，沿胸壁表面分离胸大肌与深面的胸小肌，充分暴露胸壁。在胸壁表面设计3×3cm大小的创面，创面内含至少2根肋骨。沿标记线切除胸壁，包括肋骨、肋间肌、壁层胸膜等，出血点烧灼止血，形成缺损面积3×3cm的胸壁缺损创面。7. Modeling of full-thickness chest wall defect: cut the skin along the incision line to expose the pectoralis major muscle, cut the pectoralis major muscle along the direction of the pectoralis major muscle, separate the pectoralis major muscle and the deep pectoralis minor muscle along the surface of the chest wall, fully Expose the chest wall. A 3 × 3 cm wound was designed on the surface of the chest wall, and the wound contained at least 2 ribs. The chest wall, including ribs, intercostal muscles, parietal pleura, etc., was excised along the marked line, and the bleeding point was cauterized to stop bleeding to form a chest wall defect wound with a defect area of 3 × 3 cm.
8、根据缺损面积，将rASCs膜片结合POC-PLA电纺丝补片复合体剪成约4×4cm大小，注意将补片四角修剪成钝圆。将细胞膜片侧朝向胸腔，用4-0缝线将补片固定于创面相邻的胸骨后缝合补片与创缘，注意严密封闭胸腔，缝合过程中注意不要损伤肺脏。分层缝合伤口。检查胸腔引流无液体和气体流出后，拔除引流管。8. According to the defect area, cut the rASCs membrane sheet combined with the POC-PLA electrospinning patch complex into a size of about 4 × 4 cm, and pay attention to trim the four corners of the patch into a blunt circle. With the side of the cell membrane facing the thoracic cavity, use 4-0 suture to fix the mesh to the retrosternum adjacent to the wound and suture the mesh and the wound edge. Pay attention to seal the thoracic cavity tightly, and be careful not to damage the lungs during the suture process. The wound is sutured in layers. After checking that the chest drain is free of fluid and gas, the drainage tube is removed.
9、伤口用酒精消毒，胶布固定纱布包扎。9. The wound is disinfected with alcohol, and the tape is fixed with gauze.
10、术后6h开始给予饮食。10. Start to give diet 6 hours after operation.
11、术后常规注射青霉素钠，一天一次，一次40万u，连续7天。11. Routine injection of penicillin sodium, once a day, 400,000 u once a day, for 7 consecutive days.
12、每组兔术后7天打开外敷料进行观察，胸壁切口拆线。12. The external dressings were opened for observation 7 days after the operation of the rabbits in each group, and the sutures were removed from the chest wall incision.
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments, and those skilled in the art can make various equivalents without departing from the spirit of the present invention. Modifications or substitutions of these equivalent modifications or substitutions are all included within the scope defined by the claims of the present application.
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| CN201610631267.XACN106139254B (en) | 2016-08-04 | 2016-08-04 | A kind of ASCs combination POC-PLA electrostatic spinning sticking patch complex and its preparation method and application |
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