技术领域technical field
本发明属于高分子材料和生物医用材料技术领域，涉及一种纳米纤维膜与干细胞层层叠加的人工组织工程皮肤及其制备方法。The invention belongs to the technical field of polymer materials and biomedical materials, and relates to an artificial tissue engineered skin with nanofibrous membranes and stem cells superimposed layer by layer and a preparation method thereof.
背景技术Background technique
目前，大规模烧伤等人类皮肤组织损伤都是通过自体或异体皮肤移植的方法来治疗，这不仅造成供皮区新的创伤缺陷，而且经常受到供皮来源的限制。为解决这一技术难题，随着高分子生物材料的兴起，利用组织工程技术构建模仿细胞外基质的皮肤支架来治疗皮肤损伤成为了不错的选择。但至今没有真正理想的皮肤替代物得以应用。At present, human skin tissue injuries such as large-scale burns are treated by autologous or allogeneic skin transplantation, which not only causes new wound defects in the donor area, but is often limited by the source of skin donors. To solve this technical problem, with the rise of polymer biomaterials, it has become a good choice to use tissue engineering technology to construct skin scaffolds that mimic extracellular matrix to treat skin injuries. But so far no really ideal skin substitute has been applied.
理想的人工皮肤需要具备柔软、舒适、透气透湿的特点，能够与创面有良好的贴合，同时又需要有一定的延展性、韧性和机械强度。另外，人工皮肤需要提供一个良好的材料-细胞界面，以利于自身上皮细胞的长入、促进新生皮肤附属器的再生、抵御细菌入侵，以起到促进创面愈合、皮肤功能再生的目的。现有技术中多采用水凝胶与高分子复合膜作为人工皮肤。但壳聚糖、透明质酸钠、硫酸软骨素、硫酸皮肤素等水凝胶存在制成的人工皮强度不够，透气性差的问题，而且随着用量增加，有抑制成纤维细胞生长的作用。此外，聚氨酯、硅橡胶、聚乙二醇或对苯二甲酸乙二醇酯等医用高分子材料虽然可改善人工皮肤的强度问题，但是亲水性不佳，且体内降解速度缓慢、存留时间较久甚至不降解，造成异物残留，引起无菌性炎症，影响自身细胞在创面的定植和生长，而且由于这些材料不能参与进行生理代谢，往往只能用做外层敷料。故而利用支架与细胞的相互作用设计一种工艺简单，易于量产，容易保存，生物相容性好，应用便捷，价格低廉的人工组织工程皮肤，对于快速、高效修复创面具有重要意义。The ideal artificial skin needs to be soft, comfortable, breathable and moisture-permeable, able to fit well with the wound surface, and at the same time need to have certain ductility, toughness and mechanical strength. In addition, artificial skin needs to provide a good material-cell interface to facilitate the growth of its own epithelial cells, promote the regeneration of new skin appendages, and resist bacterial invasion, so as to promote wound healing and skin function regeneration. In the prior art, hydrogel and polymer composite membranes are mostly used as artificial skin. However, hydrogels such as chitosan, sodium hyaluronate, chondroitin sulfate, and dermatan sulfate have the problems of insufficient artificial skin strength and poor air permeability, and as the dosage increases, they can inhibit the growth of fibroblasts. In addition, although medical polymer materials such as polyurethane, silicone rubber, polyethylene glycol or ethylene terephthalate can improve the strength of artificial skin, they have poor hydrophilicity, slow degradation rate in vivo, and long retention time. If it does not degrade for a long time, it will cause foreign matter residue, cause aseptic inflammation, and affect the colonization and growth of its own cells on the wound. Moreover, because these materials cannot participate in physiological metabolism, they can only be used as outer dressings. Therefore, using the interaction between scaffolds and cells to design an artificial tissue-engineered skin with simple process, easy mass production, easy storage, good biocompatibility, convenient application, and low price is of great significance for rapid and efficient wound repair.
干细胞是一种具有自我更新能力和多系分化潜能的原始细胞，是细胞移植组织再生及修复的理想细胞。虽然胚胎干细胞是最原始的及最有分化潜能的干细胞，但目前来源十分有限。而经过高表达干细胞因子诱导的干细胞也称iPS细胞，其虽然具有诸多胚胎干细胞的特性，但因制备方法复杂，还涉及到使用病毒载体等安全性问题，目前临床应用还存在很多瓶颈。间充质干细胞(Mesenchymal stem cells,MSC)是存在于人体组织中具有多系分化潜能的一种亚全能干细胞群，如骨髓干细胞、脂肪干细胞、脐带血干细胞等。脂肪干细胞来源丰富，比如可以取材于身体上多余的脂肪，游离方法简便，具有自我更新及多潜能分化能力，是一种理想的组织再生和修复材料。Stem cells are primitive cells with self-renewal ability and multilineage differentiation potential, and are ideal cells for tissue regeneration and repair after cell transplantation. Although embryonic stem cells are the most primitive stem cells with the most differentiation potential, their sources are currently very limited. Stem cells induced by high expression of stem cell factors are also called iPS cells. Although they have many characteristics of embryonic stem cells, there are still many bottlenecks in clinical application due to complicated preparation methods and safety issues such as the use of viral vectors. Mesenchymal stem cells (MSCs) are a subpotent stem cell population with multilineage differentiation potential existing in human tissues, such as bone marrow stem cells, fat stem cells, and umbilical cord blood stem cells. Adipose stem cells are rich in sources. For example, they can be obtained from excess fat in the body. The method of dissociation is simple, and they have the ability of self-renewal and multipotential differentiation. They are an ideal material for tissue regeneration and repair.
目前已有异种脱细胞基质材料用于临床。因为经过脱细胞处理，材料内不存在异种细胞，这样将避免了人体对异种材料产生的免疫反应。但因为没有细胞成分，脱细胞基质材料因此是缺乏生物活性的材料，其对创伤、组织缺损等的修复作用有限。间充质干细胞大量存在于组织中，如来源于脂肪组织的脂肪干细胞等。但是目前干细胞用于组织器官修复的细胞治疗存在诸多问题，虽然有临床应用报道，临床效果不确定。特别是用于外表创伤修复、或者损伤组织或器官的替代，如果没有细胞外基质材料的支撑，干细胞很难发挥作用。At present, xenogeneic acellular matrix materials have been used clinically. Because of the decellularization process, there are no foreign cells in the material, which will avoid the immune response of the human body to the foreign material. However, because there are no cellular components, the acellular matrix material is a material that lacks biological activity, and its repair effect on wounds and tissue defects is limited. Mesenchymal stem cells exist in large numbers in tissues, such as adipose stem cells derived from adipose tissue. However, there are many problems in the current cell therapy of stem cells for tissue and organ repair. Although there are clinical application reports, the clinical effect is uncertain. Especially for the repair of superficial wounds, or the replacement of damaged tissues or organs, it is difficult for stem cells to function without the support of extracellular matrix materials.
尽管目前有很多研究者研制开发了多种人工组织工程皮肤，多数基质为胶原凝胶或海绵，目前并没有任何通过纯天然高聚物来制备纳米纤维膜并结合层层自组装技术应用于组织工程皮肤的文献，申请号为“CN201410423055.3”与“201610353226.9”的专利分别报道了一种用于促进心肌组织再生和干细胞监测的壳聚糖-丝素蛋白复合纳米纤维多功能补片的制备方法与一种静电纺高壳聚糖含量的抗菌伤口敷料的制备方法，但并没有进行创面愈合效果的相关研究。申请号为“201610499353.X”与“201611008057.1”的专利分别报道了一种微纳米复合双层皮肤支架及其制备方法与一种柔性人工皮肤及其制备方法，但并无显著改善细胞在组织内分布与创面愈合促进效果的相关研究。Although many researchers have developed a variety of artificial tissue-engineered skins, most of which are collagen gels or sponges, there is currently no nanofiber membrane prepared from pure natural polymers combined with layer-by-layer self-assembly technology for tissue In the literature on engineered skin, the patents with application numbers "CN201410423055.3" and "201610353226.9" respectively reported the preparation of a chitosan-silk fibroin composite nanofiber multifunctional patch for promoting myocardial tissue regeneration and stem cell monitoring The method is related to the preparation of an electrospun antibacterial wound dressing with high chitosan content, but there is no related research on the wound healing effect. The patents with the application numbers "201610499353.X" and "201611008057.1" respectively reported a micro-nano composite double-layer skin scaffold and its preparation method, and a flexible artificial skin and its preparation method, but did not significantly improve the cells in the tissue. Research on the distribution and promoting effect of wound healing.
发明内容Contents of the invention
本发明的目的在于提供一种纳米纤维膜与干细胞层层叠加的人工组织工程皮肤及其制备方法，其由纳米纤维膜和干细胞两部分交替组装复合而成，可用于各种组织的再生和修复。本发明是通过以下技术方案来实现：The purpose of the present invention is to provide an artificial tissue engineered skin with nanofibrous membranes and stem cells stacked layer by layer and its preparation method, which is composed of nanofibrous membranes and stem cells alternately assembled and compounded, and can be used for regeneration and repair of various tissues . The present invention is achieved through the following technical solutions:
一种纳米纤维膜与干细胞层层叠加的人工组织工程皮肤，包括多层干细胞层和多层多孔的具有三维结构的纳米纤维膜，干细胞层与纳米纤维膜交替层叠。An artificial tissue engineered skin in which nanofibrous membranes and stem cells are laminated layer by layer, comprising multiple layers of stem cell layers and multilayer porous nanofiber membranes with a three-dimensional structure, wherein the stem cell layers and the nanofiber membranes are alternately laminated.
其中，所述交替层叠，指的是干细胞层与纳米纤维膜层层叠加，两者通过相互间隔的顺序叠加，使得干细胞层的两侧为纳米纤维膜，纳米纤维膜的两侧为干细胞层。Wherein, the alternate stacking means that the stem cell layer and the nanofiber membrane are superimposed layer by layer, and the two are superimposed in a sequence of mutual intervals, so that both sides of the stem cell layer are nanofiber membranes, and both sides of the nanofiber membrane are stem cell layers.
优选地，多层纳米纤维膜层叠形成纳米纤维支架；干细胞层中的干细胞作为种子细胞分布在纳米纤维膜表面。Preferably, multiple layers of nanofiber membranes are stacked to form a nanofiber scaffold; stem cells in the stem cell layer are distributed on the surface of the nanofiber membrane as seed cells.
优选地，所述干细胞为脂肪干细胞。Preferably, the stem cells are fat stem cells.
优选地，所述纳米纤维膜由聚己内酯和β-环糊精制备而成。Preferably, the nanofiber membrane is prepared from polycaprolactone and β-cyclodextrin.
优选地，β-环糊精中包络有布洛芬。Preferably, ibuprofen is encapsulated in β-cyclodextrin.
优选地，纳米纤维膜的透氧率为50～60％，纳米纤维膜中的纳米纤维的直径为200～600nm。Preferably, the oxygen permeability of the nanofiber membrane is 50-60%, and the diameter of the nanofibers in the nanofiber membrane is 200-600 nm.
所述的纳米纤维膜与干细胞层层叠加的人工组织工程皮肤的制备方法，包括步骤：The preparation method of the artificial tissue engineered skin in which the nanofibrous membrane and stem cells are stacked layer by layer comprises the steps of:
1)制备干细胞；1) preparing stem cells;
2)将干细胞与纳米纤维膜进行层层自组装共培养；2) Co-cultivate stem cells and nanofibrous membranes for layer-by-layer self-assembly;
其中，步骤2)包括步骤：Wherein, step 2) comprises steps:
21)以培养皿作为接收器，通过静电纺丝技术制备纳米纤维膜；21) Using a petri dish as a receiver, prepare a nanofiber membrane by electrospinning technology;
22)将干细胞接种在培养皿中；22) Seeding the stem cells in a culture dish;
23)重复步骤21)和22)，直至完成设定的接种层数；23) Repeat steps 21) and 22) until the set number of inoculation layers is completed;
24)在培养皿中，将纳米纤维膜与干细胞进行共培养。24) In a petri dish, the nanofibrous membrane is co-cultured with the stem cells.
优选地，在步骤21)中，在超净工作台中，通过静电纺丝技术制备纳米纤维膜；其中，静电纺丝过程中施加的直流电压为10～35kV；静电纺丝过程中针头与培养皿的距离为5～20cm；静电纺丝过程中注射器由注射泵以0.5～2.0mL/h的速度驱动；静电纺丝过程中环境温度为5～35℃；静电纺丝过程中环境相对湿度为20～80％；静电纺丝的时间为1～5min。Preferably, in step 21), in the ultra-clean workbench, the nanofiber membrane is prepared by electrospinning technology; wherein, the DC voltage applied during the electrospinning process is 10-35kV; The distance is 5-20cm; the syringe is driven by the syringe pump at a speed of 0.5-2.0mL/h during the electrospinning process; the ambient temperature during the electrospinning process is 5-35°C; the relative humidity of the environment during the electrospinning process is 20 ～80%; the time of electrospinning is 1～5min.
优选地，在步骤22)中，相对于纳米纤维膜的面积，干细胞接种的数量为1×104～2×104个细胞/cm2。Preferably, in step 22), relative to the area of the nanofibrous membrane, the seeding quantity of stem cells is 1×104 -2×104 cells/cm2 .
优选地，还包括步骤:3)将步骤2)制备的人工组织工程皮肤经过杀菌后密封包装，冷冻保藏。Preferably, it also includes the step: 3) sterilizing the artificial tissue engineered skin prepared in step 2), sealing and packaging, and freezing and preserving.
与现有技术相比，本发明具有以下有益的技术效果：Compared with the prior art, the present invention has the following beneficial technical effects:
本发明提供的纳米纤维膜与干细胞层层叠加的人工组织工程皮肤，其包括多层纳米纤维膜，纳米纤维膜多孔且具有三维结构，为细胞的黏附和生长提供合适的表面形态，利于细胞的粘附与生长；干细胞可以分泌各种细胞因子，促进细胞的增殖，有助于组织的再生和修复；干细胞分成多层，与纳米纤维膜交替层叠，这使得干细胞在整个人工组织工程皮肤中的分布更为均匀。动物实验证明，该人工组织工程皮肤可以显著改善细胞在组织内分布，促进创面愈合。如此，该人工组织工程皮肤可用于各种组织的再生和修复，特别是创伤愈合、减少疤痕形成、皮肤再生等。The artificial tissue engineered skin provided by the present invention is composed of nanofiber membranes and stem cells layer by layer, which includes multi-layer nanofiber membranes. The nanofiber membranes are porous and have a three-dimensional structure, which provides suitable surface morphology for cell adhesion and growth, and is beneficial to cell growth. Adhesion and growth; stem cells can secrete various cytokines, promote cell proliferation, and contribute to tissue regeneration and repair; stem cells are divided into multiple layers, which are alternately laminated with nanofibrous membranes, which makes stem cells in the entire artificial tissue engineering skin more evenly distributed. Animal experiments have proved that the artificial tissue engineered skin can significantly improve the distribution of cells in the tissue and promote wound healing. In this way, the artificial tissue engineered skin can be used for regeneration and repair of various tissues, especially wound healing, reduction of scar formation, skin regeneration and the like.
附图说明Description of drawings
图1为为本发明利用脂肪干细胞与聚己内酯-β-CD纳米纤维膜制备人工组织工程皮肤的过程示意图。Fig. 1 is a schematic diagram of the process of preparing artificial tissue-engineered skin by using adipose-derived stem cells and polycaprolactone-β-CD nanofibrous membrane in the present invention.
图2-1、图2-2和图2-3为为本发明制备的纳米纤维膜的微观结构(场发射扫描电镜)，其中图2-1放大2000倍，图2-2放大7000倍，图2-3放大15000倍。Fig. 2-1, Fig. 2-2 and Fig. 2-3 are the microstructure (field emission scanning electron microscope) of the nanofibrous membrane prepared for the present invention, wherein Fig. 2-1 enlarges 2000 times, Fig. 2-2 enlarges 7000 times, Figure 2-3 is magnified 15,000 times.
图3-1、图3-2、图3-3、图3-4、图3-5、图3-6为脂肪干细胞接种于聚己内酯-β-CD单层纳米纤维膜后的扫描电镜图，分别为接种后1，3，5，7，9，11天。Figure 3-1, Figure 3-2, Figure 3-3, Figure 3-4, Figure 3-5, Figure 3-6 are scans of adipose-derived stem cells seeded on polycaprolactone-β-CD monolayer nanofiber membrane Electron micrographs, respectively 1, 3, 5, 7, 9, 11 days after inoculation.
图4-1为单层纳米纤维膜-脂肪干细胞复合物的染色结果(红色：纳米纤维；蓝色：DAPI)，该图显示，细胞只分布于单层纳米纤维膜表面(一侧)。Figure 4-1 is the staining result of the single-layer nanofiber membrane-adipocyte stem cell complex (red: nanofiber; blue: DAPI), which shows that the cells are only distributed on the surface of the single-layer nanofiber membrane (one side).
图4-2为多层LBL制备的人工组织工程皮肤的染色结果(红色：纳米纤维；蓝色：DAPI)，该图显示，经过层层纳米纤维膜-脂肪干细胞叠加之后，细胞可均匀分布于纳米纤维膜表面与层间整个结构中，细胞密度较单层纤维膜接种可明显改善。Figure 4-2 is the staining result of artificial tissue engineered skin prepared by multi-layer LBL (red: nanofiber; blue: DAPI), which shows that after superposition of layers of nanofiber membrane-adipocyte stem cells, the cells can be evenly distributed in the In the entire structure between the nanofibrous membrane surface and the interlayer, the cell density can be significantly improved compared with the single-layer fiber membrane seeding.
图5-1、图5-2、图5-3为本发明的人工组织工程皮肤的创面愈合试验结果。结果显示，不同处理方式对于创面愈合得效果存在明显差异，其中，图5-1为对照组(常规纱布覆盖组)，图5-2为单层纳米纤维膜-脂肪干细胞复合物覆盖组，图5-3为人工组织工程皮肤覆盖组。Fig. 5-1, Fig. 5-2, Fig. 5-3 are the wound healing test results of the artificial tissue-engineered skin of the present invention. The results showed that there were significant differences in the effect of different treatment methods on wound healing. Among them, Figure 5-1 is the control group (conventional gauze covering group), Figure 5-2 is the single-layer nanofiber membrane-adipocyte stem cell composite covering group, Figure 5-1 5-3 is the artificial tissue engineering skin covering group.
图6-1、图6-2为本发明的人工组织工程皮肤覆盖创面至完全愈合后的HE染色结果，其中图6-1对照组(未处理组)；图6-2人工组织工程皮肤组。Fig. 6-1 and Fig. 6-2 are the HE staining results after the artificial tissue engineered skin of the present invention covers the wound until it is completely healed, wherein Fig. 6-1 is the control group (untreated group); Fig. 6-2 is the artificial tissue engineered skin group .
图6-3、图6-4为本发明的人工组织工程皮肤覆盖创面至完全愈合后的Masson染色结果，其中，图6-3为单层纤维膜-脂肪干细胞复合物组；图6-3为人工组织工程皮肤组。Figure 6-3 and Figure 6-4 are the Masson staining results after the artificial tissue engineered skin of the present invention covers the wound until it is completely healed, wherein Figure 6-3 is the single-layer fibrous membrane-adipocyte stem cell complex group; Figure 6-3 Engineered skin groups for artificial tissue.
具体实施方式Detailed ways
下面通过具体实施例对本发明的技术方案做进一步说明，其目的在于帮助更好的理解本发明的内容，但这些具体实施方案不以任何方式限制本发明的保护范围。The technical solutions of the present invention are further described below through specific examples, the purpose of which is to help better understand the content of the present invention, but these specific embodiments do not limit the protection scope of the present invention in any way.
本发明的目的是克服现有技术的不足，提供一种利用脂肪干细胞作为种子细胞与纳米纤维支架层层叠加的人工组织工程皮肤及其制备方法。将静电纺丝技术、高分子络合技术与纤维-细胞层层自组装技术相结合，制备具有良好的可塑性及适宜的力学特性、高度孔隙度的三维立体结构及生物相容性的组织工程皮肤，该纳米支架可调控生物降解速率，适合用于皮肤再生组织工程。The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide an artificial tissue engineered skin using adipose stem cells as seed cells and nanofiber scaffolds superimposed layer by layer and a preparation method thereof. Combining electrospinning technology, polymer complexing technology and fiber-cell layer-by-layer self-assembly technology to prepare tissue-engineered skin with good plasticity, suitable mechanical properties, three-dimensional structure with high porosity and biocompatibility , the nano-scaffold can regulate the biodegradation rate and is suitable for skin regeneration tissue engineering.
现有的脱细胞基质材料采用异种皮经脱细胞处理，使之成为不具抗原性的生物材料。由剩下的细胞外基质材料(主要由胶原和弹性蛋白组成)提供组织创面的覆盖，提供组织修复的支架而在临床应用中有一定的价值。但由于这种基质材料缺乏应有的和组织修复有关的细胞因子，其用途及有效性受到很大影响。大量研究表明，间充质干细胞如游离自脂肪的脂肪干细胞具有多分化潜能，可作为组织修复理想的种子细胞。间充质干细胞来源广泛，取材方便，免疫原性低，且不产生畸胎瘤，连续传代培养和冷冻保存后仍保留多向分化潜能，可作为组织修复理想的种子细胞。其中，脂肪干细胞(AD-MSC)大量存在于脂肪组织，易于扩增，旁分泌作用和分化能力好，作为医学整形的废料，还可加以回收利用，具有良好的临床应用前景。所以，本发明采用脂肪干细胞作为组织工程皮肤的种子细胞。Existing acellular matrix materials use xenogeneic skins to undergo decellularization to make them non-antigenic biological materials. The remaining extracellular matrix material (mainly composed of collagen and elastin) provides tissue wound coverage and provides a scaffold for tissue repair, which has certain value in clinical applications. However, since this matrix material lacks proper cytokines related to tissue repair, its use and effectiveness are greatly affected. A large number of studies have shown that mesenchymal stem cells, such as adipose stem cells dissociated from fat, have multi-differentiation potential and can be used as ideal seed cells for tissue repair. Mesenchymal stem cells have a wide range of sources, are convenient to obtain, have low immunogenicity, and do not produce teratomas. After continuous subculture and cryopreservation, they still retain multi-directional differentiation potential, and can be used as ideal seed cells for tissue repair. Among them, adipose stem cells (AD-MSCs) exist in a large amount in adipose tissue, are easy to expand, have good paracrine function and differentiation ability, and can be recycled as waste materials of medical plastic surgery, so they have good clinical application prospects. Therefore, the present invention uses adipose stem cells as seed cells for tissue-engineered skin.
当前，在人工组织工程皮肤支架领域，用于皮肤支架制备的材料大致分为两类：一类是天然生物衍生材料，如胶原、壳聚糖、透明质酸、羧甲基壳聚糖及丝素蛋白等。另一类是人工合成生物高分子材料，主要有聚乙交酯、聚己内酯、聚羟基烷酸酯、聚碳酸酯类等聚酯类材料；大多数皮肤支架都是运用生物材料通过静电纺丝技术来制造。虽然由静电纺丝得到的纳米纤维为细胞的黏附和生长提供合适的表面形态，利于细胞在支架上的粘附与生长，然而由静电纺丝制备的皮肤支架由于孔径太小，不利于细胞在深度方向上的迁移、增殖，且难以实现细胞在纤维支架上分布的有效调控，这限制了人工皮肤支架在皮肤损伤医学治疗领域中的应用。同时，作为干细胞生长的支架和外基质(ECM)，良好的亲水性、合适的表面电荷、适当的孔隙尺寸更适合细胞的附着生长。At present, in the field of artificial tissue engineering skin scaffolds, the materials used for the preparation of skin scaffolds are roughly divided into two categories: one is natural biologically derived materials, such as collagen, chitosan, hyaluronic acid, carboxymethyl chitosan and silk protein, etc. The other is artificially synthesized biopolymer materials, mainly polyester materials such as polyglycolide, polycaprolactone, polyhydroxyalkanoate, polycarbonate, etc.; most skin scaffolds use biomaterials through static electricity Spinning technology to manufacture. Although the nanofibers obtained by electrospinning provide a suitable surface morphology for cell adhesion and growth, which is conducive to the adhesion and growth of cells on the scaffold, the skin scaffold prepared by electrospinning is not conducive to the cell adhesion due to the small pore size. Migration and proliferation in the depth direction, and it is difficult to effectively regulate the distribution of cells on the fibrous scaffold, which limits the application of artificial skin scaffolds in the field of medical treatment of skin injuries. At the same time, as a scaffold and extracellular matrix (ECM) for stem cell growth, good hydrophilicity, suitable surface charge, and proper pore size are more suitable for cell attachment and growth.
根据以上特点，本发明通过静电纺丝技术，使聚己内酯-β-CD纳米纤维支架在纳米级水平加工为适合细胞生长分化的互相贯通的多孔三维微观结构，并可根据需要包被各种细胞因子或药物，还利用层层自组装(LBL)技术进行纤维-细胞层层叠加，从多个方面促进干细胞的附着和生长，提高干细胞治疗的效率。基于上述理由，本发明结合纳米纤维基质材料和间充质干细胞技术提供一种有生物学活性的生物材料，该生物材料可用于各种组织的再生和修复，特别是创伤愈合、减少疤痕形成、皮肤再生等。According to the above characteristics, the present invention processes the polycaprolactone-β-CD nanofiber scaffold at the nanoscale level into an interpenetrating porous three-dimensional microstructure suitable for cell growth and differentiation through electrospinning technology, and can be coated with various It also uses layer-by-layer self-assembly (LBL) technology to superimpose fiber-cell layer by layer, which promotes the attachment and growth of stem cells from multiple aspects, and improves the efficiency of stem cell therapy. Based on the above reasons, the present invention provides a biologically active biomaterial in combination with nanofiber matrix material and mesenchymal stem cell technology, which can be used for regeneration and repair of various tissues, especially wound healing, reducing scar formation, skin regeneration etc.
如图1所示，将脂肪干细胞接种于聚己内酯-β-CD纳米纤维膜，借助层层自组装技术进行纳米纤维膜-脂肪干细胞层层叠加，从而形成人工组织工程皮肤。As shown in Figure 1, the adipose stem cells were seeded on the polycaprolactone-β-CD nanofiber membrane, and the nanofibrous membrane-adipocyte stem cells were stacked layer by layer by means of layer-by-layer self-assembly technology to form artificial tissue engineered skin.
脂肪干细胞分离培养、纯化、鉴定方法Method for isolation, culture, purification and identification of adipose stem cells
实施例1、细胞分离纯化Embodiment 1, cell separation and purification
脂肪组织转入预先装有PBS的无菌培养皿中，用眼科剪剪去外包膜与明显结缔组织，用PBS清洗两次后剪碎；将剪碎的脂肪组织转入50mL离心管中，加入0.2％Ⅰ型胶原酶，置于37℃5％CO2培养箱中2小时，每30min混匀一次；将消化至糊状的组织通过250目金属筛过滤，滤液转入15mL离心管中，1500rpm 10min离心后，吸弃漂浮的脂肪块和上清液，PBS吹洗3次后离心，加入含10％FBS的DMEM培养液用吸管轻轻吹打，使之再重悬；接种于预先装有培养基的培养瓶中，置于37℃5％CO2培养箱中培养；静置24h后弃去培养基，用PBS清洗两次以去除未贴壁细胞，结缔组织与碎片，更换新鲜含10％FBS的DMEM培养液，每隔2天换液，观察。Transfer the adipose tissue into a sterile petri dish pre-filled with PBS, cut off the outer envelope and obvious connective tissue with ophthalmic scissors, wash it twice with PBS, and then shred it; transfer the shredded fat tissue into a 50mL centrifuge tube, Add 0.2% type Ⅰ collagenase, place in 37°C 5% CO2 incubator for 2 hours, and mix every 30 minutes; filter the digested tissue into a paste through a 250-mesh metal sieve, transfer the filtrate into a 15mL centrifuge tube, After centrifugation at 1500rpm for 10min, the floating fat pieces and supernatant were discarded, washed with PBS for 3 times and then centrifuged, then added to DMEM culture solution containing 10% FBS and gently blown with a pipette to resuspend it; Place the medium in a culture bottle at 37°C in a 5% CO2 incubator; discard the medium after standing for 24 hours, wash twice with PBS to remove unattached cells, connective tissue and debris, and replace with fresh 10 %FBS DMEM culture medium, change the medium every 2 days, and observe.
实施例2、脂肪干细胞鉴定Embodiment 2, adipose stem cell identification
取培养的第3代细胞，吸出培养基后，先用PBS清洗两次，加入预热的0.25％胰酶消化，之后加入含10％FBS的DMEM终止消化，轻轻吹打成单细胞悬液1000rpm条件离心5min后，弃去上清，PBS清洗两次；取10个上样管，每管加入1mL单细胞悬液，分别依次加入10μL IgG-FITC，IgG-PE，鼠抗人CD29-FITC,CD44-FITC，CD49d-FITC，CD73-FITC，CD90-FITC，CD105-FITC，CD34-PE，CD45-PE单克隆抗体工作液；室温下避光孵育20min；PBS清洗两次以去除未结合抗体，500μL PBS重悬后采用流式细胞仪检测。Take the cultured 3rd passage cells, suck out the culture medium, wash twice with PBS, add preheated 0.25% trypsin to digest, then add DMEM containing 10% FBS to stop the digestion, gently pipette into a single cell suspension After centrifugation at 1000rpm for 5min, discard the supernatant and wash twice with PBS; take 10 sample tubes, add 1mL single cell suspension to each tube, add 10μL IgG-FITC, IgG-PE, mouse anti-human CD29-FITC respectively , CD44-FITC, CD49d-FITC, CD73-FITC, CD90-FITC, CD105-FITC, CD34-PE, CD45-PE monoclonal antibody working solution; incubate at room temperature in the dark for 20 minutes; wash twice with PBS to remove unbound antibodies , resuspended in 500 μL PBS and detected by flow cytometry.
实施例3、细胞扩增Embodiment 3, cell expansion
待细胞生长至80-90％汇合后进行传代，吸出培养基，先用PBS清洗两次，然后加入0.25％胰酶，置于37℃恒温培养箱2min，随后加入含10％FBS的DMEM培养液中止消化，吸管轻轻吹打使细胞脱落，收集细胞，移入15mL离心管中。1000rpm 5min，弃上清，PBS清洗两次，加入含10％FBS的DMEM重悬细胞沉淀，按1:2密度接种于培养瓶中，加入含10％FBS的DMEM置于37℃5％CO2培养箱中继续培养，显微镜下观察细胞形态，获得扩增的脂肪干细胞。Subculture after the cells grow to 80-90% confluence, suck out the medium, wash twice with PBS, then add 0.25% trypsin, place in a constant temperature incubator at 37°C for 2 minutes, then add DMEM culture medium containing 10% FBS Stop the digestion, blow the pipette gently to dislodge the cells, collect the cells, and transfer them to a 15mL centrifuge tube. 1000rpm for 5 minutes, discard the supernatant, wash twice with PBS, add DMEM containing 10% FBS to resuspend the cell pellet, inoculate in a culture flask at a density of 1:2, add DMEM containing 10% FBS and place at 37°C with 5% CO2 The culture was continued in the incubator, and the cell morphology was observed under a microscope to obtain expanded adipose stem cells.
实施例4、一种聚己内脂-β-CD纳米纤维膜的制备方法Embodiment 4, a kind of preparation method of polycaprolactone-β-CD nanofiber membrane
在超净工作台内，将聚己内酯(PCL,Mn＝70-90KDa,Sigma AldrichCo.,USA)溶解至质量比为1:(1～3)的N,N-二甲基甲酰胺—二氯甲烷混合溶液中，配置成质量浓度为8％的聚己内酯溶液；向聚己内酯溶液中加入β-CD，使得β-CD的质量分数为0.1％，加热至50～70℃，搅拌均匀，搅拌时间为6～12小时，得聚己内酯-β-CD溶液；In the ultra-clean workbench, dissolve polycaprolactone (PCL, Mn=70-90KDa, Sigma AldrichCo., USA) to N,N-dimethylformamide with a mass ratio of 1:(1~3)— In the dichloromethane mixed solution, configure a polycaprolactone solution with a mass concentration of 8%; add β-CD to the polycaprolactone solution so that the mass fraction of β-CD is 0.1%, and heat to 50-70°C , stir evenly, and the stirring time is 6 to 12 hours to obtain a polycaprolactone-β-CD solution;
其中，β-CD中可以预先包络有布洛芬：称取摩尔质量比为1:10～1:1的β-CD与布洛芬混合物于干燥研钵中，在密封环境中向研钵中加入13％去离子水，室温研磨1～3h，将制好的包和物在真空干燥箱内35-60℃下干燥6-12h，得到β-CD/布洛芬包合物。Among them, ibuprofen can be pre-enveloped in β-CD: Weigh the mixture of β-CD and ibuprofen with a molar mass ratio of 1:10 to 1:1 in a dry mortar, and pour it into the mortar in a sealed environment. Add 13% deionized water to the mixture, grind at room temperature for 1-3 hours, and dry the prepared clathrate in a vacuum oven at 35-60° C. for 6-12 hours to obtain β-CD/ibuprofen clathrate.
在超净工作台内，将获得的聚己内酯-β-CD溶液吸入静电纺丝设备的带金属针头的塑料注射器。注射器针头与作为收集器的培养皿相距10㎝，施加的直流电压为20千伏。注射器由注射泵以1.0mL/h的速度驱动，环境温度为25℃，相对湿度40％，静电纺丝时间为3min，获得纳米纤维膜，该纳米纤维膜被接收在培养皿中。Inside the ultra-clean bench, draw the obtained polycaprolactone-β-CD solution into a plastic syringe with a metal needle of the electrospinning device. The distance between the syringe needle and the petri dish as a collector was 10 cm, and a DC voltage of 20 kV was applied. The syringe was driven by a syringe pump at a speed of 1.0 mL/h, the ambient temperature was 25 °C, the relative humidity was 40%, and the electrospinning time was 3 min to obtain a nanofibrous membrane, which was received in a petri dish.
实施例5、一种聚己内脂-β-CD纳米纤维膜的制备方法Example 5, a preparation method of polycaprolactone-β-CD nanofiber membrane
参照实施例4的方法，其中，聚己内酯-β-CD溶液中，聚己内酯的质量浓度为4％；β-CD的质量浓度为0.02％；针头与培养皿的距离为20cm，施加的直流电压为10千伏；注射器由注射泵以2.0mL/h的速度驱动，环境温度为35℃，相对湿度为20％，静电纺丝时间为5min。With reference to the method of Example 4, wherein, in the polycaprolactone-β-CD solution, the mass concentration of polycaprolactone is 4%; the mass concentration of β-CD is 0.02%; the distance between the needle and the petri dish is 20cm, The applied DC voltage was 10 kV; the syringe was driven by a syringe pump at a speed of 2.0 mL/h, the ambient temperature was 35 °C, the relative humidity was 20%, and the electrospinning time was 5 min.
实施例6、一种聚己内脂-β-CD纳米纤维膜的制备方法Example 6, a preparation method of polycaprolactone-β-CD nanofiber membrane
参照实施例4的方法，其中，聚己内酯-β-CD溶液中，聚己内酯的质量浓度为12％；β-CD的质量浓度为0.2％；针头与培养皿的距离为5cm，施加的直流电压为35千伏；注射器由注射泵以0.5m L/h的速度驱动，环境温度为5℃，相对湿度为80％，静电纺丝时间为1min。With reference to the method of Example 4, wherein, in the polycaprolactone-β-CD solution, the mass concentration of polycaprolactone is 12%; the mass concentration of β-CD is 0.2%; the distance between the needle and the petri dish is 5cm, The applied DC voltage was 35 kV; the syringe was driven by a syringe pump at a speed of 0.5 mL/h, the ambient temperature was 5 °C, the relative humidity was 80%, and the electrospinning time was 1 min.
利用层层自组装技术(LBL技术)在聚己内脂-β-CD纳米纤维膜表面交替接种脂肪干细胞进行层层三维共培养Using layer-by-layer self-assembly technology (LBL technology) to alternately inoculate adipose-derived stem cells on the surface of polycaprolactone-β-CD nanofiber membranes for layer-by-layer three-dimensional co-culture
实施例7Example 7
在超净工作台内，参照实施例4～6的方法，制备被接收在培养皿中的纳米纤维膜，其中，培养皿的直径为30mm；将实施例3获得的扩增的脂肪干细胞接种在具有纳米纤维膜的培养皿中，接种的细胞悬浮液的体积为1mL，细胞数量为1×105个细胞；然后再将该培养皿作为纳米纤维膜的接收器进行静电纺丝，静电纺丝结束后再进行脂肪干细胞接种，如此，每层接种1×105个细胞，总共接种10层。因为整个过程发生在培养基表面，细胞在组装工程中一直保持水合状态，总共有10层细胞/纳米纤维交替的层层叠加成三维结构。将培养皿放置在37℃CO2培养箱培养30分钟，然后加入DMEM/F12补充培养基；继续培养1周以形成一种层层自组装形成的人工组织工程皮肤。其中DMEM/F12培养液包括10％胎牛血清和1％青霉素/链霉素。In the ultra-clean workbench, referring to the method of Examples 4-6, prepare the nanofibrous membrane received in the culture dish, wherein the diameter of the culture dish is 30mm; inoculate the adipose stem cells obtained in Example 3 on the In a petri dish with a nanofiber membrane, the volume of the inoculated cell suspension is 1 mL, and the number of cells is1 ×105 cells; then the petri dish is used as a receiver of the nanofiber membrane for electrospinning. Inoculation of adipose-derived stem cells was performed after the end, so that each layer was inoculated with 1×105 cells, and a total of 10 layers were inoculated. Because the whole process takes place on the surface of the medium, the cells remain hydrated during the assembly process, and a total of 10 layers of cells/nanofibers are stacked alternately to form a three-dimensional structure. Place the culture dish in a 37°C CO2 incubator and incubate for 30 minutes, then add DMEM/F12 supplementary medium; continue to incubate for 1 week to form a layer-by-layer self-assembled artificial tissue-engineered skin. The DMEM/F12 culture medium includes 10% fetal bovine serum and 1% penicillin/streptomycin.
其中，若在纳米纤维膜表面仅接种一次脂肪干细胞，借助层层自组装技术层叠纳米纤维膜和脂肪干细胞，通过培养后可以形成单层纳米纤维膜-脂肪干细胞复合物。Among them, if the adipose stem cells are inoculated only once on the surface of the nanofibrous membrane, the nanofibrous membrane and adipose stem cells are stacked by layer-by-layer self-assembly technology, and a single-layer nanofibrous membrane-adipocyte stem cell complex can be formed after culture.
实施例8Example 8
参照实施例6的制备方法，其中，培养皿的直径为100mm，每层接种的脂肪干细胞的数量为1×106个细胞。Referring to the preparation method of Example 6, wherein the diameter of the culture dish is 100 mm, and the number of adipose stem cells inoculated in each layer is 1×106 cells.
实施例9Example 9
参照实施例6的制备方法，其中，培养皿的直径为100mm，每层接种的脂肪干细胞的数量为1×106个细胞，总共接种20层。Referring to the preparation method in Example 6, wherein the diameter of the culture dish is 100 mm, the number of adipose stem cells inoculated in each layer is 1×106 cells, and a total of 20 layers are inoculated.
实施例10实施例7～9所制备的层层自组装形成的人工组织工程皮肤，通过环氧乙烷杀菌后密封包装，包装袋中具有冷冻保护剂，包装后冷冻保存；包装后的工程皮肤可以冷冻在-78～82℃的冰箱中，也可以冷冻在液氮中。Example 10 The artificial tissue-engineered skin formed by layer-by-layer self-assembly prepared in Examples 7-9 is sterilized by ethylene oxide and then sealed and packaged. There is a cryoprotectant in the packaging bag, and it is frozen after packaging; It can be frozen in a refrigerator at -78 to 82°C, or in liquid nitrogen.
实施例11Example 11
将1×1cm2的聚己内酯-β-环糊精纤维膜样品进行真空喷碳处理后，采用场发射扫描电子显微镜上观察纤维形貌，加速电压为15kV。所得实验结果展示在图2-1、图2-2和图2-3中，其中，图2-1放大2000倍，图2-2放大7000倍，图2-3放大15000倍。结果显示，制备的聚己内酯-β-CD纳米纤维膜呈交叉的立体3D结构，纳米纤维为粗细均一的细丝状，直径与孔隙分布均匀，呈无序排布结构，平均纤维直径为350±72nm。After the 1×1cm2 polycaprolactone-β-cyclodextrin fiber film sample was subjected to vacuum carbon spraying treatment, the morphology of the fiber was observed on a field emission scanning electron microscope with an accelerating voltage of 15kV. The experimental results obtained are shown in Figure 2-1, Figure 2-2 and Figure 2-3, wherein Figure 2-1 is enlarged by 2000 times, Figure 2-2 is enlarged by 7000 times, and Figure 2-3 is enlarged by 15000 times. The results show that the prepared polycaprolactone-β-CD nanofiber membrane has a crossed three-dimensional 3D structure, and the nanofibers are filaments with uniform thickness, uniform distribution of diameter and pores, and a disordered arrangement structure. The average fiber diameter is 350±72nm.
实施例12Example 12
将脂肪干细胞接种于聚己内酯-β-环糊精纳米纤维膜上共培养3天后，采用戊二醛溶液在4℃下固定过夜后，用PBS清洗，经过一系列乙醇梯度脱水后进行真空冷冻干燥，干燥6h后进行真空喷碳处理，利用场发射扫描电镜观察细胞在纤维表面的生长情况。Adipose-derived stem cells were seeded on polycaprolactone-β-cyclodextrin nanofiber membranes and co-cultured for 3 days, fixed overnight at 4°C with glutaraldehyde solution, washed with PBS, dehydrated in a series of ethanol gradients, and then vacuumed Freeze-drying, vacuum carbon spraying treatment after drying for 6 hours, using field emission scanning electron microscopy to observe the growth of cells on the fiber surface.
图3-1、图3-2、图3-3、图3-4、图3-5、图3-6分别为为脂肪干细胞接种于聚己内酯-β-CD单层纳米纤维膜后1，3，5，7，9，11天的扫描电镜图。扫描结果显示，脂肪干细胞与纳米纤维膜黏附紧密，可明显观察到细胞的丝状与板状伪足伸展，细胞微丝互相缠绕或相互连接，呈网状结构，细胞部分或全部迁徙到材料孔隙内部，细胞表面及周围可见颗粒状结构，可能为细胞分泌的细胞因子或蛋白质，说明细胞生长状态良好。随着培养天数增加，部分细胞重叠呈三维生长，细胞间形成伪足识别，并形成复层(图3-6)，表明制备的纳米纤维膜具有良好的生物相容性，可显著促进脂肪干细胞的粘附与增殖。Fig. 3-1, Fig. 3-2, Fig. 3-3, Fig. 3-4, Fig. 3-5, Fig. 3-6 respectively show the adipose-derived stem cells after seeding on polycaprolactone-β-CD monolayer nanofibrous membrane Scanning electron micrographs of days 1, 3, 5, 7, 9, and 11. The scanning results show that the adipose stem cells adhere tightly to the nanofiber membrane, and the extension of the filaments and lamellipodia of the cells can be clearly observed, and the cell microfilaments are intertwined or connected with each other, forming a network structure, and some or all of the cells migrate to the pores of the material Inside, granular structures can be seen on and around the cell surface, which may be cytokines or proteins secreted by the cells, indicating that the cells are in good growth condition. As the number of days of culture increases, some cells overlap and grow three-dimensionally, form pseudopodia recognition between cells, and form stratified layers (Figure 3-6), indicating that the prepared nanofibrous membrane has good biocompatibility and can significantly promote adipose-derived stem cells. adhesion and proliferation.
实施例13Example 13
将脂肪干细胞接种于单层聚己内酯-β-环糊精纳米纤维膜，共培养3天，用PBS清洗3次；采用4％甲醛溶液(溶于PBS)固定30min，干燥5min；随后采用PBS清洗3次；并用0.5％TRITON X-100渗透20min，随后用PBS进行清洗；采用DAPI对细胞核在室温下进行染色10min(稀释度：1:1000)，随后采用罗丹明对纳米纤维进行染色5min。用PBS清洗来移除未结合的染色液，封片进行共聚焦观察。实验结果展示在图4-1中，其中，红色部分为纳米纤维，蓝色部分为DAPI；该图显示，细胞只分布于单层纳米纤维膜表面(即只在纳米纤维膜一侧)。Adipose-derived stem cells were inoculated on a single-layer polycaprolactone-β-cyclodextrin nanofiber membrane, co-cultured for 3 days, washed 3 times with PBS; fixed with 4% formaldehyde solution (dissolved in PBS) for 30 min, and dried for 5 min; Wash 3 times with PBS; infiltrate with 0.5% TRITON X-100 for 20 minutes, then wash with PBS; use DAPI to stain the cell nucleus at room temperature for 10 minutes (dilution: 1:1000), and then use rhodamine to stain the nanofibers for 5 minutes . Wash with PBS to remove unbound staining solution, and seal the slides for confocal observation. The experimental results are shown in Figure 4-1, where the red part is the nanofiber, and the blue part is DAPI; this figure shows that the cells are only distributed on the surface of the single-layer nanofiber membrane (that is, only on one side of the nanofiber membrane).
将脂肪干细胞与聚己内酯-β-环糊精纳米纤维膜采用层层自组装技术进行交替叠加后，共培养3天，用PBS清洗3次；采用4％甲醛溶液(溶于PBS)固定30min，干燥5min；随后采用PBS清洗3次；并用0.5％TRITON X-100渗透20min，随后用PBS进行清洗；采用DAPI对细胞核在室温下进行染色10min(稀释度：1:1000)，随后采用罗丹明对纳米纤维进行染色5min。用PBS清洗来移除未结合的染色液，封片进行共聚焦观察。实验结果展示在图4-2中，其中，红色部分为纳米纤维，蓝色部分为DAPI；该图显示，经过层层纳米纤维-脂肪干细胞叠加之后，细胞可均匀分布于纳米纤维膜表面与层间整个结构中，细胞密度较单层纤维接种可明显改善。Adipose-derived stem cells and polycaprolactone-β-cyclodextrin nanofibrous membranes were stacked alternately by layer-by-layer self-assembly technology, co-cultured for 3 days, washed 3 times with PBS; fixed with 4% formaldehyde solution (dissolved in PBS) 30min, dry for 5min; then wash 3 times with PBS; permeate with 0.5% TRITON X-100 for 20min, then wash with PBS; Brightly dye the nanofibers for 5 min. Wash with PBS to remove unbound staining solution, and seal the slides for confocal observation. The experimental results are shown in Figure 4-2, in which the red part is the nanofiber, and the blue part is DAPI; this figure shows that after superposition of layers of nanofiber-adipocyte stem cells, the cells can be evenly distributed on the surface and layer of the nanofiber membrane In the entire structure, the cell density can be significantly improved compared with single-layer fiber seeding.
实施例14Example 14
选择健康、成年、清洁级SD大鼠，雌雄各半，体质量200-250g，SPF级，由第四军医大学动物中心提供。按随机数字表法将其随机分为模型组与对照组，每组4只，共三组(对照组(常规纱布覆盖组)，单层纳米纤维膜-脂肪干细胞复合物覆盖组，人工组织工程皮肤覆盖组)，每组大鼠术前12h禁食。大鼠进行腹腔注射麻醉，剃净鼠背部毛，碘伏消毒背部皮肤，铺无菌巾单。在背部用模具量取2cm直径的皮肤范围，周边用实线标记，切除实线标记范围内的全层皮肤至深筋膜层，形成全层皮肤缺损创面。Select healthy, adult, clean-grade SD rats, half male and half male, body weight 200-250 g, SPF grade, provided by the Animal Center of Fourth Military Medical University. According to the random number table method, they were randomly divided into model group and control group, with 4 rats in each group, a total of three groups (control group (conventional gauze covering group), single-layer nanofiber membrane-adipocyte stem cell composite covering group, artificial tissue engineering Skin-covered group), and the rats in each group were fasted for 12 hours before operation. The rats were anesthetized by intraperitoneal injection, the hair on the back of the rat was shaved, the skin on the back was disinfected with povidone iodine, and a sterile towel was spread. Use a mold to measure the skin area with a diameter of 2 cm on the back, mark the periphery with a solid line, and resect the full-thickness skin to the deep fascia layer within the range marked by the solid line to form a full-thickness skin defect wound.
对照组，单层油纱覆盖创面；In the control group, a single layer of oil gauze was used to cover the wound;
单层纳米纤维-脂肪干细胞复合物覆盖组：单层纳米纤维膜-脂肪干细胞复合物覆盖创面；Single-layer nanofiber-adipocyte stem cell composite covering group: single-layer nanofiber membrane-adipocyte stem cell composite covers the wound;
人工组织工程皮肤组：人工组织工程皮肤覆盖组Artificial Tissue Engineering Skin Group: Artificial Tissue Engineering Skin Covering Group
依照分组，将覆盖材料按照创面大小剪裁，覆盖创面，取材时间以各组动物的创面愈合时间为准。在创面形成后在不同周期对创面愈合情况进行实时监控，利用图像分析软件分析创面治疗前后照片，愈合率大于90％即判定为愈合。实验结果展示在图5-1、图5-2、图5-3中；其中，图5-1为对照组(常规纱布覆盖组)，图5-2为单层纳米纤维-脂肪干细胞复合物覆盖组，图5-3为人工组织工程皮肤覆盖组。结果显示，不同处理方式对于创面愈合得效果存在明显差异，经本发明制备的人工组织工程皮肤处理的创面愈合后再上皮化程度更高，说明该材料对于创面愈合的促进效果最佳。According to the grouping, the covering material was cut according to the size of the wound to cover the wound, and the collection time was based on the wound healing time of animals in each group. After the wound was formed, the wound healing was monitored in real time at different periods, and the photos before and after the wound treatment were analyzed using image analysis software. If the healing rate was greater than 90%, it was judged to be healed. The experimental results are shown in Figure 5-1, Figure 5-2, and Figure 5-3; among them, Figure 5-1 is the control group (conventional gauze-covered group), and Figure 5-2 is the single-layer nanofiber-adipocyte stem cell composite Covering group, Figure 5-3 is the artificial tissue engineering skin covering group. The results show that there are obvious differences in the effects of different treatment methods on wound healing, and the re-epithelialization degree of wound healing treated by the artificial tissue engineered skin prepared by the present invention is higher, indicating that the material has the best promoting effect on wound healing.
实施例15Example 15
在实施例14的基础上，在创面完全愈合后取新生组织(带创缘部分)，经石蜡包埋、切片，采用常规苏木精-伊红(HE)染色检测；检测结果展示在图6-1、图6-2中，其中，图6-1对照组(未处理组)，图6-2人工组织工程皮肤组。与对照组(无基底膜，表-真皮层分离)相比，人工组织工程皮肤组未观察到明显的表-真皮层分离，且可观察到真皮层有部分新生血管。On the basis of Example 14, new tissues (with wound margins) were taken after the wound surface was completely healed, embedded in paraffin, sectioned, and detected by conventional hematoxylin-eosin (HE) staining; the detection results are shown in Figure 6 -1. In Figure 6-2, wherein, Figure 6-1 is the control group (untreated group), and Figure 6-2 is the artificial tissue engineered skin group. Compared with the control group (no basement membrane, epidermis separation), the artificial tissue engineered skin group did not observe obvious epidermis-dermis separation, and some new blood vessels in the dermis could be observed.
在创面完全愈合后取新生组织(带创缘部分)，经石蜡包埋、切片后进行烘片、脱蜡、梯度入水，漂洗后，依次采用R1，R2，R3，R4液进行染色(碧云天染色试剂盒)，中性树胶封片，采用荧光显微镜观察组织内胶原纤维、血管与其他皮肤结构。实验结果展示在图6-3、图6-4中，其中，图6-3为对照组(未处理组)；图6-4为人工组织工程皮肤组。Masson染色结果显示，本发明制备的人工组织工程皮肤具有优良的体内降解性能，并且在材料植入后有新生血管生成。实验结果证实：本发明制备的人工组织工程皮肤在同一植入周期内具有更好的体内降解性能，且纤维内部可观察到新生血管生成。After the wound was completely healed, the new tissue (the part with the wound edge) was taken, embedded in paraffin, sliced, dried, dewaxed, soaked in water gradiently, rinsed, and stained with R1, R2, R3, R4 in sequence (Biyuntian staining kit), mounted with neutral gum, and observed collagen fibers, blood vessels and other skin structures in the tissue with a fluorescence microscope. The experimental results are shown in Figure 6-3 and Figure 6-4, wherein Figure 6-3 is the control group (untreated group); Figure 6-4 is the artificial tissue engineered skin group. The results of Masson staining show that the artificial tissue engineered skin prepared by the present invention has excellent degradation performance in vivo, and new blood vessels can be formed after the material is implanted. Experimental results confirm that the artificial tissue engineered skin prepared by the present invention has better in vivo degradation performance within the same implantation period, and new blood vessels can be observed inside the fibers.
Formhals于1934年首次研发出一种借助高压静电场激发聚合物的带电射流，使射流固化得到超细结构的纳米纤维，通过该方法制备的纳米纤维具备超细纤维直径、较大比表面积和三维立体结构等独特优势，这使其逐渐成为皮肤创面修复领域的研究热点。本发明采用静电纺丝技术，其原因在于:首先，静电纺丝技术所构建的组织工程支架在结构上具有模拟细胞外基质(extracellularmatrix,ECM)的作用，可以促进成纤维细胞与角质形成细胞的粘附、增殖与迁移；其次，本发明的静电纺丝溶液中以具有良好生物相容性的聚己内酯为原料，这种纳米纤维支架可显著加速创面愈合。第三、β-CD(β-环糊精)具有外缘亲水而内腔疏水的特殊结构，可通过形成主客体包络物从而提高包和物的稳定性与药物缓释能力。本发明在此基础上，利用纳米纤维膜-细胞层层自组装的新技术进行纳米纤维层层叠加以实现细胞组装构成三维结构，这种三维结构可以良好控制细胞分布，允许共培养多种细胞，并且在物理上分隔各种细胞却不影响其物质交换。申请者所在实验室及课题组王红军最近建立起一种新兴的通过纳米纤维层层叠加实现细胞组装的技术，并已经成功将这种技术运用于构建三维组织(US 20080112998A1)。在这种层层叠加细胞组装中，细胞被添加到纳米纤维膜之间。每层细胞层的细胞种类和密度以及每层纤维膜的组成和厚度都可以根据需要调节。Formhals first developed a charged jet in 1934 with the help of a high-voltage electrostatic field to excite the polymer to solidify the jet to obtain nanofibers with ultrafine structures. The nanofibers prepared by this method have ultrafine fiber diameters, large specific surface areas and three-dimensional properties. The unique advantages such as three-dimensional structure make it gradually become a research hotspot in the field of skin wound repair. The present invention adopts electrospinning technology, and its reason is: at first, the tissue engineering support that electrospinning technology is constructed has the effect of simulating extracellular matrix (extracellular matrix, ECM) in structure, can promote fibroblast and keratinocyte. Adhesion, proliferation and migration; secondly, polycaprolactone with good biocompatibility is used as raw material in the electrospinning solution of the present invention, and this nanofibrous scaffold can significantly accelerate wound healing. Third, β-CD (β-cyclodextrin) has a special structure in which the outer edge is hydrophilic and the inner cavity is hydrophobic, which can improve the stability and drug release ability of the inclusion by forming a host-guest inclusion. On this basis, the present invention uses the new technology of nanofiber membrane-cell self-assembly to superimpose nanofiber layers to realize cell assembly to form a three-dimensional structure. This three-dimensional structure can well control the distribution of cells and allow co-cultivation of various cells. And physically separate various cells without affecting their material exchange. Wang Hongjun, the applicant's laboratory and research group, has recently established a new technology for cell assembly through layer-by-layer stacking of nanofibers, and has successfully applied this technology to construct three-dimensional tissues (US 20080112998A1). In this layer-by-layer cell assembly, cells are added between nanofibrous membranes. The cell type and density of each cell layer and the composition and thickness of each fibrous membrane can be adjusted as needed.
与现有技术相比，本发明具有以下有益效果:Compared with prior art, the present invention has following beneficial effect:
1)脂肪干细胞来源丰富，获取方法简单；1) Adipose stem cells are rich in sources and easy to obtain;
2)脂肪干细胞具有多分化潜能性，能分化成各种胚层的细胞，如上皮细胞、神经细胞、肌肉细胞、成纤维细胞等；2) Adipose stem cells have multi-differentiation potential and can differentiate into cells of various germ layers, such as epithelial cells, nerve cells, muscle cells, fibroblasts, etc.;
3)脂肪干细胞能分泌多种细胞因子，如血管生长因子、表皮生长因子(EGF)、血小板衍生生长因子(PDGF)等，可以促进组织细胞再生；3) Adipose stem cells can secrete a variety of cytokines, such as vascular growth factor, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), etc., which can promote tissue cell regeneration;
4)因脂肪干细胞取自自身，因此不具抗原性；4) Since the adipose stem cells are taken from themselves, they are not antigenic;
5)静电纺丝聚己内酯组织工程支架在结构上具有模拟细胞外基质(extracellular matrix,ECM)的作用，可以促进脂肪干细胞的粘附、增殖与迁移；并可显著加速创面愈合；5) The electrospun polycaprolactone tissue engineering scaffold has the function of simulating the extracellular matrix (ECM) in structure, which can promote the adhesion, proliferation and migration of adipose-derived stem cells; and can significantly accelerate wound healing;
6)β-CD具有外缘亲水而内腔疏水的特殊结构，可通过形成主客体包络物从而提高包和物的稳定性与药物缓释能力；6) β-CD has a special structure in which the outer edge is hydrophilic and the inner cavity is hydrophobic, which can improve the stability and drug release ability of the inclusion by forming a host-guest inclusion;
7)利用纳米纤维-细胞层层自组装的新技术进行纳米纤维层层叠加以实现细胞组装构成三维结构组织。这种三维结构可以良好控制细胞分布，允许共培养多种细胞，并且在物理上分隔各种细胞却不影响其物质交换。7) Using the new technology of nanofiber-cell layer-by-layer self-assembly to stack nanofibers layer by layer to realize cell assembly to form a three-dimensional structural organization. This three-dimensional structure can well control the distribution of cells, allow co-cultivation of multiple cells, and physically separate various cells without affecting their material exchange.
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| CN201710577134.3ACN107349475B (en) | 2017-07-14 | 2017-07-14 | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell |
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| CN201710577134.3ACN107349475B (en) | 2017-07-14 | 2017-07-14 | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell |
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| CN201710577134.3AActiveCN107349475B (en) | 2017-07-14 | 2017-07-14 | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell |
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