技术领域technical field
本发明涉及医学技术领域，尤其涉及一种微/纳米仿生骨膜材料及其制备方法。The invention relates to the field of medical technology, in particular to a micro/nano bionic periosteum material and a preparation method thereof.
背景技术Background technique
目前，由于创伤、肿瘤、骨病导致的骨折延迟愈合甚至不愈合，在骨科临床工作中仍旧是一个很大的挑战。以往人们过多的关注于骨缺损本身，而忽略了骨膜的重要性以及其在骨科疾病发生发展时易损性，因而大量的治疗方案始终围绕于骨移植物来研究和开展，这包括异体骨、自体骨、以及各种各样的组织工程植骨材料。尽管目前外科技术以及植骨材料研究飞速发展，但是仍有10％的骨折病人，会发展成为骨延迟愈合甚至不愈合，由此带来的残疾问题导致了沉重的社会-经济负担。At present, delayed union or even nonunion of fractures caused by trauma, tumors, and bone diseases is still a big challenge in orthopedic clinical work. In the past, people paid too much attention to the bone defect itself, while ignoring the importance of the periosteum and its vulnerability in the development of orthopedic diseases. Therefore, a large number of treatment options have been researched and carried out around bone grafts, including allografts. , autologous bone, and a variety of tissue engineering bone graft materials. Despite the rapid development of surgical techniques and bone grafting materials, 10% of fracture patients will develop delayed union or even nonunion, and the resulting disability will lead to a heavy social-economic burden.
作为正常骨组织的重要组成成分，骨膜在骨的发育和损伤的修复中起着至关重的作用。骨膜为包覆于骨组织周围的一种薄而坚韧的结缔组织膜，可以分为内、外两层，但二层并无截然分界。其中外层称为“纤维层”，其细胞成分较少，主要含有胶原和弹力纤维，以及丰富的血管网，对骨膜组织起结构性支撑以及提供血液供应的作用。内层即“生发层”，紧贴骨表面，富含前成骨细胞以及具有多向分化潜能的间充质干细胞，这些细胞在必要时可以分化为成骨细胞或者成软骨细胞。因此，骨膜复杂的内在结构和多种功能的需要造成了损伤后修复与重建的困难。As an important component of normal bone tissue, periosteum plays a crucial role in bone development and repair of damage. The periosteum is a thin and tough connective tissue membrane covering the bone tissue. It can be divided into inner and outer layers, but there is no clear boundary between the two layers. The outer layer is called the "fibrous layer", which has less cellular components and mainly contains collagen and elastic fibers, as well as a rich vascular network, which provides structural support for the periosteal tissue and provides blood supply. The inner layer, the "germinal layer", adheres to the bone surface and is rich in preosteoblasts and mesenchymal stem cells with multilineage differentiation potential, which can differentiate into osteoblasts or chondrocytes when necessary. Therefore, the complex internal structure and multiple functions of the periosteum make it difficult to repair and reconstruct after injury.
骨膜起源于中胚层的间充质，由间充质细胞和无定形基质发育而来。在胚胎发育过程中，间充质先浓缩聚集形成软骨雏形和一层包绕其上的纤维膜。这层纤维膜被称作软骨膜，其富含间充质细胞。随着软骨膜的血管化，间充质细胞增生并向成骨细胞方向分化，逐渐形成一层包绕软骨雏形的被称作骨领的原始骨组织。随着骨领的形成，软骨膜改称为骨膜。然后，由于骨膜组织的血管和成骨细胞的浸润生长，软骨细胞开始退变和凋亡，最终软骨基质钙化。这一现象即软骨内成骨的过程。外层的骨膜则以膜内成骨的方式继续成骨，骨膜成骨细胞分泌骨基质和纤维，外周基质逐渐矿化，最终成骨细胞被包埋在钙化基质中变为骨细胞。显然的，骨膜的发育过程是一个协调而有序的过程。The periosteum originates from the mesenchyme of the mesoderm and develops from mesenchymal cells and an amorphous matrix. During embryonic development, the mesenchyme first condenses and aggregates to form a cartilage prototype and a fibrous membrane surrounding it. This fibrous membrane is called the perichondrium and is rich in mesenchymal cells. With the vascularization of the perichondrium, mesenchymal cells proliferate and differentiate toward osteoblasts, gradually forming a layer of primitive bone tissue called the bone collar surrounding the cartilage prototype. With the formation of the bony collar, the perichondrium is renamed the periosteum. Then, due to the infiltration and growth of blood vessels and osteoblasts in the periosteal tissue, chondrocytes begin to degenerate and undergo apoptosis, and eventually the cartilage matrix becomes calcified. This phenomenon is the process of endochondral bone formation. The outer periosteum continues to form bone in the way of intramembranous osteogenesis. Periosteal osteoblasts secrete bone matrix and fibers, and the peripheral matrix gradually mineralizes. Finally, osteoblasts are embedded in the calcified matrix and become bone cells. Obviously, the development of periosteum is a coordinated and orderly process.
目前，许多不同的生物和高分子材料组合的策略已被研究用于骨膜重建，包括脱细胞真皮骨膜，水凝胶复合细胞骨膜，PLGA微图案纳米片，hMSCs和HUVECs复合细胞片层等。这些骨膜组织工程材料确实证实了其具有良好的骨再生效果，但是不可避免的外源性细胞存活困难、免疫排斥反应，潜在的病毒感染风险等缺点都使这些研究的实际运用受限。更重要的是，这类骨膜材料都是基于骨膜的某些结构特点和功能，例如宏观结构仿生骨膜、微观结构仿生骨膜以及血管化骨膜，而没有真正从结构和功能的角度解决骨膜再生问题，组织再生过程和自然生长过程也不匹配。Currently, strategies combining many different biological and polymer materials have been investigated for periosteum reconstruction, including acellular dermal periosteum, hydrogel composite cell periosteum, PLGA micropatterned nanosheets, hMSCs and HUVECs composite cell sheet, etc. These periosteum tissue engineering materials have indeed confirmed that they have good bone regeneration effects, but the inevitable difficulties in the survival of exogenous cells, immune rejection, and potential virus infection risks have limited the practical application of these studies. More importantly, this kind of periosteum materials are based on certain structural characteristics and functions of periosteum, such as macroscopic bionic periosteum, microscopic bionic periosteum, and vascularized periosteum, but do not really solve the problem of periosteum regeneration from the perspective of structure and function. The tissue regeneration process also does not match the natural growth process.
发明内容Contents of the invention
鉴于此，本发明的目的在于提供一种微/纳米仿生骨膜材料及其制备方法。本发明提供的微/纳米纤维仿生骨膜材料具有良好的生物相容性和生物活性，体外能有效促进间充质干细胞的黏附、增殖和成骨分化以及内皮细胞的血管形成。本发明以一种外源性的仿生骨膜材料替代天然骨膜纤维层，能隔绝外周软组织，减少瘢痕组织生成，促进早期血管生成、促进间充质细胞增殖分化形成内源性的生发层，最终通过外源-内源相结合的途径模拟骨膜的发育过程完成骨膜的修复，并通过骨膜固有的成骨机制来使骨缺损快速而均匀的修复。In view of this, the object of the present invention is to provide a micro/nano bionic periosteum material and a preparation method thereof. The micro/nanofiber bionic periosteum material provided by the present invention has good biocompatibility and bioactivity, and can effectively promote the adhesion, proliferation, osteogenic differentiation of mesenchymal stem cells and angiogenesis of endothelial cells in vitro. The present invention replaces the natural periosteum fiber layer with an exogenous bionic periosteum material, which can isolate peripheral soft tissue, reduce scar tissue formation, promote early angiogenesis, promote the proliferation and differentiation of mesenchymal cells to form an endogenous germinal layer, and finally pass The combination of exogenous and endogenous approaches simulates the development of periosteum to complete the repair of periosteum, and repairs bone defects quickly and uniformly through the inherent osteogenesis mechanism of periosteum.
为了实现本发明的目的，本发明采用如下技术方案：In order to realize the purpose of the present invention, the present invention adopts following technical scheme:
本发明将微溶胶(Micro-sol)静电纺丝技术和纳米胶原纤维自组装技术结合，以形成具有核-鞘结构的PLLA微米纤维膜和致密沉积在所述PLLA纤维膜内的纳米胶原纤维构成的微/纳米纤维结构为特点，从而构建了一种可血管化的微/纳米仿生骨膜材料。The present invention combines micro-sol (Micro-sol) electrospinning technology and nano-collagen fiber self-assembly technology to form a PLLA micron fiber membrane with a core-sheath structure and nano-collagen fibers densely deposited in the PLLA fiber membrane Characterized by the micro/nano fiber structure, a vascularized micro/nano biomimetic periosteum material was constructed.
一些实施方案中，所述胶原纤维为I型胶原蛋白自组装形成的纳米纤维。In some embodiments, the collagen fibers are nanofibers formed by self-assembly of type I collagen.
本发明还提供了仿生骨膜材料的制备方法，包括以下步骤：The present invention also provides a preparation method of the bionic periosteum material, comprising the following steps:
步骤1：以PLLA为外鞘，VEGF、HA为核芯，制备包裹VEGF-HA的PLLA微米级纤维膜；Step 1: Using PLLA as the outer sheath and VEGF and HA as the core, prepare a PLLA micron-sized fiber membrane wrapped with VEGF-HA;
步骤2：将中性I型胶原蛋白溶液滴加到所述PLLA微米级纤维膜上，I型胶原蛋白经自组装形成纳米纤维，所述纳米纤维致密地沉积在所述PLLA微米级纤维膜的表面和孔隙获得微/纳米纤维结构，即所述微/纳米仿生骨膜材料。Step 2: Add the neutral type I collagen solution dropwise onto the PLLA micron-scale fiber membrane, the type I collagen self-assembles to form nanofibers, and the nanofibers are densely deposited on the PLLA micron-scale fiber membrane The surface and pores obtain a micro/nano fiber structure, that is, the micro/nano biomimetic periosteum material.
本发明中，在具体实施例以及效果实验考察中，步骤1制备的包裹VEGF、HA的PLLA微米级纤维膜，将其命名为微溶胶(MS)纤维膜，即MS纤维膜；将本发明所述的微/纳米仿生骨膜材料命名为MS-Col纤维膜。In the present invention, in specific embodiment and effect experiment investigation, the PLLA micron-scale fiber membrane of wrapping VEGF, HA prepared in step 1 is named microsol (MS) fiber membrane, i.e. MS fiber membrane; The micro/nano biomimetic periosteum material named MS-Col fibrous membrane.
本发明采用了一种改良的超稳定的乳液电纺技术，将其命名为微溶胶电纺技术。该技术中，首先VEGF被包裹于透明质酸(HA)水溶液内，之后将VEGF-HA水相分散于纺丝液有机相中形成微溶胶颗粒。稳定性是乳液法静电纺丝的关键属性。相较于传统的乳液体系不稳定、微粒易聚集的特点，HA微溶胶乳液体系中，DLS粒径分析显示微溶胶粒子能稳定的分散于DCM有机相中，并且其粒径大小在2小时内不会发生明显改变。这说明该乳液中HA微溶胶粒子不会很快发生聚集，其稳定性超越传统乳液体系。再通过静电纺丝技术，由于水油两相的粘度梯度差异，微溶胶颗粒会包裹于PLLA纤维中形成核-鞘结构，即包裹VEGF-HA核芯的PLLA纤维膜，从而使纤维层具有血管化的能力。相比于，传统的乳液电纺丝以及同轴电纺技术，微溶胶电纺采用HA溶胶为载体，设备简单、操作简易、药物装载效率高，药物释放时间长且可控。然后，在上述PLLA纤维膜上进行I型胶原蛋白自组装。通过对PLLA纤维膜的充分水化，调定至中性的胶原溶液可以有效通过水分子的动力学因素渗透进疏水的PLLA纤维膜、组装形成的纳米纤维致密的沉积于上述PLLA纤维的表面与孔隙内构成微/纳米纤维结构，最终获得本发明所述的仿生骨膜材料。本发明中称其为MS-Col微/纳米纤维膜或MS-Col纤维膜。实验表明，本发明仿生骨膜不仅增强了纤维膜的物理性能，隔绝骨损伤部位与外周软组织，也增强了生物相容性，构建了适合间充质细胞生存的微环境，促进间充质细胞的黏附、增殖以及成骨分化构成生发层，以及内皮细胞的血管化，从而实现骨膜的结构和功能的再生。最后，在大鼠颅骨骨膜骨缺损模型中，仿生骨膜显示了独特的骨膜修复，血管化、骨再生以及阻止疤痕组织的侵入的能力。The present invention adopts an improved ultra-stable emulsion electrospinning technology, which is named microsol electrospinning technology. In this technology, VEGF is first encapsulated in hyaluronic acid (HA) aqueous solution, and then the VEGF-HA aqueous phase is dispersed in the organic phase of the spinning solution to form microsol particles. Stability is a key attribute of emulsion electrospinning. Compared with the traditional emulsion system which is unstable and the particles are easy to aggregate, in the HA microsol emulsion system, the DLS particle size analysis shows that the microsol particles can be stably dispersed in the DCM organic phase, and the particle size is within 2 hours. No noticeable change will occur. This shows that the HA microsol particles in the emulsion will not aggregate quickly, and its stability exceeds that of the traditional emulsion system. Then, through electrospinning technology, due to the difference in the viscosity gradient of the water and oil phases, the microsol particles will be wrapped in the PLLA fibers to form a core-sheath structure, that is, the PLLA fiber membrane that wraps the VEGF-HA core, so that the fiber layer has blood vessels. ability to transform. Compared with the traditional emulsion electrospinning and coaxial electrospinning technologies, microsol electrospinning uses HA sol as the carrier, which has simple equipment, easy operation, high drug loading efficiency, and long and controllable drug release time. Then, type I collagen self-assembly was performed on the above-mentioned PLLA fibrous membrane. Through sufficient hydration of the PLLA fiber membrane, the collagen solution adjusted to neutrality can effectively penetrate into the hydrophobic PLLA fiber membrane through the kinetic factors of water molecules, and the assembled nanofibers are densely deposited on the surface of the above-mentioned PLLA fiber and The micro/nano fiber structure is formed in the pores, and finally the bionic periosteum material described in the present invention is obtained. In the present invention, it is called MS-Col micro/nano fiber membrane or MS-Col fiber membrane. Experiments show that the bionic periosteum of the present invention not only enhances the physical properties of the fibrous membrane, isolates the bone injury site from the peripheral soft tissue, but also enhances biocompatibility, constructs a microenvironment suitable for the survival of mesenchymal cells, and promotes the growth of mesenchymal cells. Adhesion, proliferation, and osteogenic differentiation constitute the germinal layer, as well as vascularization of endothelial cells, resulting in structural and functional regeneration of the periosteum. Finally, in a rat calvarial periosteal bone defect model, the biomimetic periosteum showed unique capabilities for periosteal repair, vascularization, bone regeneration, and resistance to scar tissue invasion.
本发明中，步骤1中所述包裹VEGF-HA的PLLA微米级纤维膜的制备方法为：In the present invention, the preparation method of the PLLA micron-scale fiber membrane wrapping VEGF-HA described in step 1 is:
以含有VEGF和HA的水溶液为水相，以第一有机溶剂和表面活性剂的混合物为油相，制得油包水微乳；向所述油包水微乳中依次加入PLLA和第二有机溶剂，获得微溶胶纺丝溶液；将所述微溶胶纺丝溶液进行微溶胶静电纺丝，得包裹VEGF-HA的PLLA微米级纤维膜。The aqueous solution containing VEGF and HA is used as the water phase, and the mixture of the first organic solvent and surfactant is used as the oil phase to prepare a water-in-oil microemulsion; PLLA and the second organic solvent to obtain a microsol spinning solution; the microsol spinning solution is subjected to microsol electrospinning to obtain a PLLA micron fiber membrane wrapped with VEGF-HA.
一些实施方案中，步骤1制备所述包裹有VEGF-HA的PLLA纤维膜的方法中，以mg/g计，所述水相与所述油相的质量比为60:4.01；In some embodiments, in the method for preparing the PLLA fiber membrane wrapped with VEGF-HA in step 1, in mg/g, the mass ratio of the water phase to the oil phase is 60:4.01;
所述水相中，VEGF与HA的质量比为1:600；In the water phase, the mass ratio of VEGF to HA is 1:600;
所述油相中，第一溶剂和表面活性剂的质量比为4:0.01。In the oil phase, the mass ratio of the first solvent to the surfactant is 4:0.01.
一些实施方案中，所述第一溶剂、PLLA和第二有机溶剂的质量比为4：0.5:2。In some embodiments, the mass ratio of the first solvent, PLLA and the second organic solvent is 4:0.5:2.
一些具体实施例中，所述水相由以下方法制得：In some specific embodiments, the aqueous phase is prepared by the following method:
取10ul 100ug/ml(按10mg计算)VEGF水溶液与50mg 1.2％wt水溶液按照质量比为1:5的比例混合，充分混匀，得含有VEGF和HA的水溶液，即所述水相。Take 10ul of 100ug/ml (calculated as 10mg) VEGF aqueous solution and 50mg of 1.2%wt aqueous solution according to the mass ratio of 1:5 and mix thoroughly to obtain an aqueous solution containing VEGF and HA, that is, the aqueous phase.
一些实施方案中，所述第一有机溶剂为二氯甲烷；所述表面活性剂为Span-80；所述第二有机溶剂为N,N-二甲基甲酰胺。In some embodiments, the first organic solvent is dichloromethane; the surfactant is Span-80; and the second organic solvent is N,N-dimethylformamide.
本发明步骤1中，所述静电纺丝步骤为：将微溶胶纺丝溶液加入到连有钝头钢针的注射器中，将一个金属夹子夹在针的头端处，金属夹连接到直流高压电源供应器上。将铝箔附在自制圆形滚筒式上并连接负极来收集电纺丝纤维，获得包裹VEGF-HA的PLLA纤维膜。In step 1 of the present invention, the electrospinning step is: adding the microsol spinning solution into a syringe connected with a blunt steel needle, clamping a metal clip at the head end of the needle, and connecting the metal clip to a DC high voltage on the power supply. The aluminum foil was attached to a self-made circular drum and connected to the negative electrode to collect the electrospun fibers, and the PLLA fiber membrane wrapped with VEGF-HA was obtained.
在一些具体实施例中，静电纺丝采用的注射器为连有钝头钢针的10ml的注射器，注射针的内径为9mm。纺丝时，针头距收集器约15cm，推注速率为60ul/min，电压约15-20kV。In some specific embodiments, the syringe used for electrospinning is a 10ml syringe connected with a blunt steel needle, and the inner diameter of the injection needle is 9mm. When spinning, the needle is about 15cm away from the collector, the injection rate is 60ul/min, and the voltage is about 15-20kV.
本发明中，步骤2中所述中性I型胶原蛋白溶液由以下方法制得：In the present invention, the neutral type I collagen solution described in step 2 is prepared by the following method:
取Col-I溶解在0.1M的醋酸溶液中形成终浓度为3-3.5mg/ml的胶原溶液，然后用0.1M NaOH溶液调定PH至7.0，最后加入10×PBS缓冲液，使PBS占总溶液的体积百分比为1：6，获得所述中性I型胶原蛋白溶液。Dissolve Col-I in 0.1M acetic acid solution to form a collagen solution with a final concentration of 3-3.5mg/ml, then adjust the pH to 7.0 with 0.1M NaOH solution, and finally add 10×PBS buffer to make PBS account for the total The volume percentage of the solution is 1:6 to obtain the neutral type I collagen solution.
在一些具体实施例中，所述中性I型胶原蛋白溶液由以下方法制备得到：In some specific embodiments, the neutral type I collagen solution is prepared by the following method:
将冻干的Col-I溶解在0.1M的醋酸溶液中形成终浓度为3mg/ml的胶原溶液，储存于4℃冰箱中备用。使用时，用0.1M的NaOH将pH调至7.0，再加入10xPBS缓冲液使PBS占总溶液体积比为1：6，最终获得所述的中性I型胶原蛋白溶液。The lyophilized Col-I was dissolved in 0.1 M acetic acid solution to form a collagen solution with a final concentration of 3 mg/ml, and stored in a refrigerator at 4°C for use. When in use, adjust the pH to 7.0 with 0.1M NaOH, then add 10xPBS buffer solution to make the volume ratio of PBS to the total solution 1:6, and finally obtain the neutral type I collagen solution.
本发明步骤2中在将中性I型胶原蛋白溶液与所述包裹VEGF-HA的PLLA纤维膜复合之前还包括对PLLA纤维膜进行预处理的步骤，所述预处理为：取所述包裹VEGF-HA的PLLA纤维膜浸泡在75％的乙醇中充分水化、灭菌30min，然后用去离子水冲洗。In step 2 of the present invention, before the neutral type I collagen solution is compounded with the PLLA fiber membrane wrapping VEGF-HA, the step of pretreating the PLLA fiber membrane is also included, and the pretreatment is: taking the wrapping VEGF - The PLLA fiber membrane of HA was fully hydrated by soaking in 75% ethanol, sterilized for 30 minutes, and then rinsed with deionized water.
本发明中，步骤2中将中性I型胶原蛋白溶液与所述包裹VEGF-HA的PLLA纤维膜复合的方式为滴加，即将中性I型胶原蛋白溶液均匀滴加至包裹VEGF-HA的PLLA纤维膜上。In the present invention, in step 2, the method of compounding the neutral type I collagen solution with the PLLA fiber membrane wrapping VEGF-HA is dripping, that is, the neutral type I collagen solution is evenly added dropwise to the wrapping VEGF-HA. PLLA fiber membrane.
本发明中，所述自组装反应的温度为37℃，时间为30-60min。在一些具体实施例中，自组装反应的温度为37℃，时间为30min。In the present invention, the temperature of the self-assembly reaction is 37° C., and the time is 30-60 minutes. In some specific embodiments, the temperature of the self-assembly reaction is 37° C., and the time is 30 minutes.
本发明还提供了由本发明以上制备方法制得的微/纳米仿生骨膜材料。The present invention also provides the micro/nano bionic periosteum material prepared by the above preparation method of the present invention.
本发明提供一种微/纳米仿生骨膜材料及其制备方法，所述制备方法包括：步骤1：以PLLA为外鞘，VEGF、HA为核芯，制备包裹VEGF-HA的PLLA微米级纤维膜；The invention provides a micro/nano bionic periosteum material and a preparation method thereof. The preparation method comprises: step 1: using PLLA as the outer sheath, VEGF and HA as the core, and preparing a PLLA micron-scale fiber membrane wrapped with VEGF-HA;
步骤2：将I型胶原蛋白溶液滴加到所述PLLA微米级纤维膜上，I型胶原蛋白经自组装形成纳米纤维，所述纳米纤维致密地沉积在所述PLLA微米级纤维膜的表面和孔隙获得微/纳米纤维结构，即所述微/纳米仿生骨膜材料。本发明制得的微/纳米仿生骨膜材料体外考察中具有良好的物理性能和生物相容性，能促进间充质细胞的黏附、增殖以及成骨分化形成内源性的生发层，并促进内皮细胞的血管分化。在动物体内考察中，本发明以一种外源性的仿生骨膜材料替代天然骨膜纤维层，隔绝外周软组织，减少瘢痕组织生成，促进早期血管生成、促进间充质细胞增殖分化形成内源性的生发层，最终通过外源-内源相结合的途径模拟骨膜的发育过程完成骨膜的修复，并通过骨膜固有的成骨机制来使骨缺损快速而均匀的修复。Step 2: the type I collagen solution is added dropwise on the PLLA micron-scale fiber membrane, and the type I collagen forms nanofibers through self-assembly, and the nanofibers are densely deposited on the surface of the PLLA micron-scale fiber membrane and The pores acquire a micro/nanofibrous structure, that is, the micro/nano biomimetic periosteum material. The micro/nano bionic periosteum material prepared by the present invention has good physical properties and biocompatibility in vitro, can promote the adhesion, proliferation and osteogenic differentiation of mesenchymal cells to form endogenous germinal layers, and promote endothelial Vascular differentiation of cells. In animal investigations, the present invention replaces the natural periosteum fiber layer with an exogenous bionic periosteum material, isolates peripheral soft tissue, reduces scar tissue formation, promotes early angiogenesis, and promotes the proliferation and differentiation of mesenchymal cells to form endogenous The germinal layer finally completes the repair of the periosteum by simulating the development process of the periosteum through the combination of exogenous and endogenous sources, and repairs the bone defect quickly and uniformly through the inherent osteogenesis mechanism of the periosteum.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.
图1示本发明仿生骨膜材料制备的流程示意图，其中1-A为包裹VEGF的微溶胶静电纺丝纤维膜的制备，1-B为MS-Col纤维膜结构的构建；1-C为胶原自组装的过程，1-D为仿生骨膜大体观察和应用示意图；Fig. 1 shows the schematic flow chart of the preparation of bionic periosteum material of the present invention, wherein 1-A is the preparation of the microsol electrospun fiber membrane that wraps VEGF, and 1-B is the construction of MS-Col fiber membrane structure; The assembly process, 1-D is the general observation and application diagram of the bionic periosteum;
图2示各组样品的微观观察结果，其中，2-A为样品的SEM图，2-B为样品的TEM图，2-C和2-D分别为PLLA和MS电纺纤维直径分布图，2-E代表PLLA和MS电纺纤维直径统计学无差异；Figure 2 shows the microscopic observation results of each group of samples, where 2-A is the SEM image of the sample, 2-B is the TEM image of the sample, 2-C and 2-D are the diameter distribution graphs of PLLA and MS electrospun fibers, respectively, 2-E represents that there is no statistical difference in the diameter of PLLA and MS electrospun fibers;
图3示各组样品的理化性能测试结果，其中，3-A为微溶胶乳液体系中的HA粒子粒径分析；3-B各组水接触角的测量结果；3-C胶原自组装后的流变测试；3-D为各组材料红外图谱测试；3-E为各组材料力学拉伸测试；3-F为仿生骨膜组(MS-Col纤维膜)VEGF释放曲线；Fig. 3 shows the physical and chemical performance test results of each group of samples, wherein, 3-A is the particle size analysis of HA particles in the microsol emulsion system; 3-B is the measurement result of water contact angle of each group; 3-C collagen self-assembly Rheological test; 3-D is the infrared spectrum test of each group of materials; 3-E is the mechanical tensile test of each group of materials; 3-F is the VEGF release curve of the bionic periosteum group (MS-Col fiber membrane);
图4示间充质干细胞(BMSCs)在不同组别纤维膜和Control组上培养3天后的细胞染色图，其中活细胞被染为绿色荧光，死细胞被染为红色荧光；Figure 4 shows the cell staining diagram of mesenchymal stem cells (BMSCs) after being cultured on different groups of fiber membranes and the Control group for 3 days, wherein live cells are stained with green fluorescence, and dead cells are stained with red fluorescence;
图5示细胞培养1、3、5、7天后的增值结果，其中，统计学分析：ns表示无统计学差异；**表示MS-Col和MS组间有差异，P<0.01；##，Control组和MS-Col组有组间差异，P<0.01；Figure 5 shows the results of cell proliferation after 1, 3, 5, and 7 days of cell culture. Among them, statistical analysis: ns means no statistical difference; ** means there is a difference between MS-Col and MS groups, P<0.01; ##, There was a difference between the Control group and the MS-Col group, P<0.01;
图6示不同纤维膜上的细胞粘附形态；Figure 6 shows the cell adhesion morphology on different fiber membranes;
图7示使用Integrin抗体对细胞进行免疫荧光染色的结果与半定量分析，其中，7-A为Integrinβ1免疫荧光染色结果，7-B位使用Image J软件对荧光强度进行半定量的结果；统计学分析：ns，无统计学差异；**，P<0.01；Figure 7 shows the results and semi-quantitative analysis of immunofluorescent staining of cells using Integrin antibodies, wherein 7-A is the result of immunofluorescent staining of Integrinβ1, and 7-B is the result of semi-quantitative fluorescence intensity using Image J software; statistics Analysis: ns, no statistical difference; **, P<0.01;
图8示使用Vinculin抗体对细胞进行免疫荧光染色的结果与半定量分析，8-A为Vinculin免疫荧光染色，8-B为使用Image J软件对荧光强度进行半定量；统计学分析:ns，无统计学差异；**，P<0.01；Figure 8 shows the results and semi-quantitative analysis of immunofluorescent staining of cells using Vinculin antibody, 8-A is Vinculin immunofluorescent staining, and 8-B is semi-quantitative use of Image J software for fluorescence intensity; statistical analysis: ns, no Statistical difference; **, P<0.01;
图9示ALP活性染色和定量分析结果，其中9-A为ALP活性染色结果，9-B为ALP活性定量测定；统计学分析:ns，无统计学意义；**，MS-Col和Control组有差异，P<0.01；##，MS-Col和MS组有差异，P<0.01；Fig. 9 shows ALP activity staining and quantitative analysis result, and wherein 9-A is ALP activity staining result, and 9-B is ALP activity quantitative determination; Statistical analysis: ns, no statistical significance; **, MS-Col and Control group There are differences, P<0.01; ##, there are differences between MS-Col and MS groups, P<0.01;
图10示茜素红染色和钙结节含量测定结果，其中10-A为茜素红染色结果,10-B为钙结节含量测定，统计学分析：ns，无统计学意义；**，MS-Col和Control组有差异，P<0.01；##，MS-Col和MS组有差异，P<0.01；Figure 10 shows the results of alizarin red staining and calcium nodule content determination, wherein 10-A is the result of alizarin red staining, 10-B is the determination of calcium nodule content, statistical analysis: ns, no statistical significance; **, There is a difference between MS-Col and Control group, P<0.01; ##, there is a difference between MS-Col and MS group, P<0.01;
图11示PLLA纤维膜和MS纤维膜HUVECs成管实验结果，其中11-A、11-B分别为内皮细胞在培养3h、6h之后形成网络状血管结构图，11-C和11-D分别为每个高倍镜视野管状结构节点数量、长度的定量分析；统计学分析：*，P<0.05；**，P<0.01；##，P<0.01；Figure 11 shows the results of PLLA fibrous membrane and MS fibrous membrane HUVECs tube formation experiments, in which 11-A and 11-B are diagrams of the network-like vascular structure formed by endothelial cells after cultured for 3 hours and 6 hours respectively, and 11-C and 11-D are respectively Quantitative analysis of the number and length of tubular structure nodes in each high-power field of view; statistical analysis: *, P<0.05; **, P<0.01; ##, P<0.01;
图12示仿生骨膜的体内评估结果，其中12-A为不同组别颅骨缺损区域micro-CT的3D重建图片，12-B为骨量在总体积中的占比(BV/TV)；统计学分析：ns，无统计学差异；**，MS-Col和PLLA-Col组有差异，P<0.01；&,PLLA-Col和MS group有差异，P<0.05；#,PLLA和Control组有差异，P<0.05；Figure 12 shows the in vivo evaluation results of bionic periosteum, in which 12-A is the 3D reconstruction picture of micro-CT in the skull defect area of different groups, and 12-B is the proportion of bone volume in the total volume (BV/TV); statistics Analysis: ns, no statistical difference; **, difference between MS-Col and PLLA-Col group, P<0.01; &, difference between PLLA-Col and MS group, P<0.05; #, difference between PLLA and Control group , P<0.05;
图13示4周和8周的CD31免疫组化分析染色图，图中黄色箭头表示新生血管；Figure 13 shows CD31 immunohistochemical analysis staining diagrams at 4 weeks and 8 weeks, and the yellow arrows in the figure indicate new blood vessels;
图14示8周时的periostin免疫组化分析，黑色箭头表示集中、线性排列的periostin。Figure 14 shows the immunohistochemical analysis of periostin at 8 weeks, and the black arrows indicate concentrated and linear arrangement of periostin.
具体实施方式Detailed ways
本发明公开了一种微/纳米仿生骨膜材料及其制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a micro/nano bionic periosteum material and a preparation method thereof. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
对所公开的实施例的说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。The disclosed embodiments are described to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
本发明采用的试材皆为普通市售品，皆可于市场购得。The test materials used in the present invention are all common commercially available products, which can be purchased in the market.
下面结合实施例，进一步阐述本发明：Below in conjunction with embodiment, further set forth the present invention:
实施例1本发明仿生骨膜材料(MS-Col微/纳米纤维膜)的制备Embodiment 1 Preparation of bionic periosteum material (MS-Col micro/nano fiber membrane) of the present invention
(1)微溶胶(MS)纺丝溶液的制备(1) Preparation of microsol (MS) spinning solution
将12mg的透明质酸钠粉末溶解于988mg去离子水中，制备质量分数为1.2wt％的HA水溶液。将预先分装好的10ul(10mg)VEGF水溶液(100ug/ml)与50mg 1.2wt％HA水溶液充分混匀，最终形成HA质量百分含量为1％wt的HA-VEGF的水溶液60mg。然后，在4g二氯甲烷(DCM)溶液中加入0.01gSpan-80，搅拌均匀后，再上述配置完成的1％HA-VEGF水溶液缓慢滴入，高速搅拌约30min，形成均一稳定的，包裹VEGF的HA微溶胶颗粒的油包水微乳(W/O)。在此油包水微乳中加入0.5g PLLA固体颗粒，在室温下用磁力搅拌器进行搅拌溶解，直至PLLA溶解形成略粘稠均一的溶液。随后，将2g二甲基甲酰胺(DMF)溶液加入其中，用磁力搅拌器继续搅拌，获得微溶胶(MS)纺丝溶液。12 mg of sodium hyaluronate powder was dissolved in 988 mg of deionized water to prepare a HA aqueous solution with a mass fraction of 1.2 wt%. Fully mix 10ul (10mg) VEGF aqueous solution (100ug/ml) pre-packed with 50mg 1.2wt% HA aqueous solution to form 60mg HA-VEGF aqueous solution with HA mass percentage of 1%wt. Then, add 0.01g of Span-80 to 4g of dichloromethane (DCM) solution, stir evenly, then slowly drop in the above-mentioned 1% HA-VEGF aqueous solution, and stir at high speed for about 30min to form a uniform and stable VEGF-wrapped Water-in-oil microemulsion (W/O) of HA microsol particles. Add 0.5 g of PLLA solid particles to the water-in-oil microemulsion, and stir and dissolve with a magnetic stirrer at room temperature until the PLLA dissolves to form a slightly viscous and uniform solution. Subsequently, 2 g of dimethylformamide (DMF) solution was added thereto, and the stirring was continued with a magnetic stirrer to obtain a microsol (MS) spinning solution.
(2)微溶胶静电纺丝纤维膜的制备(2) Preparation of microsol electrospun fiber membrane
将制备好的微溶胶(MS)纺丝溶液加入到连有钝头钢针的10ml的注射器中。注射针的内径为9mm。将一个金属夹子夹在针的头端处，金属夹连接到直流高压电源供应器上。将铝箔附在自制圆形滚筒式上并连接负极来收集电纺丝纤维，精密推进泵被用来控制注射速率。开始纺丝，针头距收集器约15cm，推注速率为60ul/min，电压约15-20kV，最终得到包裹VEGF-HA的PLLA微米纤维膜，即微溶胶(MS)纤维膜。The prepared microsol (MS) spinning solution was added to a 10ml syringe connected with a blunt steel needle. The inner diameter of the injection needle is 9 mm. Clip a metal clip over the tip of the needle and connect the metal clip to the DC high voltage power supply. Aluminum foil was attached to a homemade circular drum and connected to the negative electrode to collect the electrospun fibers, and a precision propulsion pump was used to control the injection rate. Start spinning, the needle is about 15cm away from the collector, the injection rate is 60ul/min, the voltage is about 15-20kV, and finally the PLLA micron fiber membrane wrapped with VEGF-HA is obtained, that is, the microsol (MS) fiber membrane.
(3)MS-Col微/纳米纤维结构的构建(3) Construction of MS-Col micro/nanofiber structure
为了使一型胶原(Col-I)能将电纺膜充分功能化，MS纤维膜先在75％的乙醇中水化和灭菌30min。用去离子水将电纺膜冲洗3遍，以完全去除残余的乙醇。将冻干的Col-I溶解在0.1M的醋酸溶液中形成终浓度为3mg/ml的胶原溶液，并储存于4℃冰箱中备用。使用时，用0.1M的NaOH将pH调至7.0，再加入10xPBS缓冲液使PBS占总溶液体积比为1：6，最终获得所述的中性I型胶原蛋白溶液。以上所有的操作都需要的冰浴上完成。将适量的中性胶原溶液滴在PLLA或MS纤维膜上，然后置于37℃的恒温箱中30min来完成胶原自组装的过程。组装完成后，用去离子水冲洗纤维膜三遍，得到MS-Col微/纳米纤维膜，即仿生骨膜材料。In order to fully functionalize the electrospun membrane with type I collagen (Col-I), the MS fiber membrane was first hydrated and sterilized in 75% ethanol for 30 min. The electrospun membrane was rinsed 3 times with deionized water to completely remove residual ethanol. The lyophilized Col-I was dissolved in 0.1 M acetic acid solution to form a collagen solution with a final concentration of 3 mg/ml, and stored in a 4°C refrigerator for future use. When in use, adjust the pH to 7.0 with 0.1M NaOH, then add 10xPBS buffer solution to make the volume ratio of PBS to the total solution 1:6, and finally obtain the neutral type I collagen solution. All the above operations need to be done on ice bath. An appropriate amount of neutral collagen solution was dropped on the PLLA or MS fiber membrane, and then placed in a thermostat at 37°C for 30 minutes to complete the collagen self-assembly process. After the assembly was completed, the fiber membrane was rinsed three times with deionized water to obtain the MS-Col micro/nano fiber membrane, namely the bionic periosteum material.
对比例1 PLLA-Col纤维膜的制备Preparation of comparative example 1 PLLA-Col fiber membrane
(1)PLLA静电纺丝液的制备(1) Preparation of PLLA electrospinning solution
首先，将预先称重好的0.5g PLLA固体颗粒加入到4g DCM溶液中，在室温下用磁力搅拌器进行搅拌溶解，直至PLLA溶解形成略粘稠均一的溶液。随后，将2g DMF溶液加入其中，用磁力搅拌器继续搅拌，即可制备出透明均一的PLLA纺丝溶液。First, add 0.5 g of PLLA solid particles weighed in advance to 4 g of DCM solution, stir and dissolve with a magnetic stirrer at room temperature until PLLA dissolves to form a slightly viscous and uniform solution. Subsequently, 2 g of DMF solution was added thereto, and the stirring was continued with a magnetic stirrer to prepare a transparent and uniform PLLA spinning solution.
(2)PLLA静电纺丝纤维膜的制备(2) Preparation of PLLA electrospun fiber membrane
将制备好的PLLA电纺丝溶液加入到连有钝头钢针的10ml的注射器中。注射针的内径为9mm。将一个金属夹子夹在针的头端处，金属夹连接到直流高压电源供应器上。将铝箔附在自制圆形滚筒式上并连接负极来收集电纺丝纤维，精密推进泵被用来控制注射速率。开始纺丝，针头距收集器约15cm，推注速率为60ul/min，电压约15-20kV，最终得到PLLA纤维膜。The prepared PLLA electrospinning solution was added into a 10ml syringe connected with a blunt steel needle. The inner diameter of the injection needle is 9 mm. Clip a metal clip over the tip of the needle and connect the metal clip to the DC high voltage power supply. Aluminum foil was attached to a homemade circular drum and connected to the negative electrode to collect the electrospun fibers, and a precision propulsion pump was used to control the injection rate. Start spinning, the needle is about 15cm away from the collector, the injection rate is 60ul/min, the voltage is about 15-20kV, and finally the PLLA fiber membrane is obtained.
将PLLA纤维膜先需要在75％的乙醇中水化和灭菌30min。用去离子水将电纺膜冲洗3遍，以完全去除残余的乙醇。将冻干的Col-I溶解在0.1M的醋酸溶液中形成终浓度为3mg/ml的胶原溶液，并储存于4℃冰箱中备用。使用时，用0.1M的NaOH将pH调至7.0，再加入10xPBS缓冲液使PBS占总溶液体积比为1：6，最终获得所述的中性I型胶原蛋白溶液。以上所有的操作都需要的冰浴上完成。最后，将样品置于37℃的恒温箱中30min来完成胶原自组装的过程。组装完成后，用去离子水冲洗纤维膜三遍，即可得到PLLA-Col微/纳米纤维膜。The PLLA fiber membrane needs to be hydrated and sterilized in 75% ethanol for 30 minutes. The electrospun membrane was rinsed 3 times with deionized water to completely remove residual ethanol. The lyophilized Col-I was dissolved in 0.1 M acetic acid solution to form a collagen solution with a final concentration of 3 mg/ml, and stored in a 4°C refrigerator for future use. When in use, adjust the pH to 7.0 with 0.1M NaOH, then add 10xPBS buffer solution to make the volume ratio of PBS to the total solution 1:6, and finally obtain the neutral type I collagen solution. All the above operations need to be done on ice bath. Finally, the sample was placed in a 37°C incubator for 30 minutes to complete the collagen self-assembly process. After the assembly is completed, rinse the fiber membrane with deionized water three times to obtain the PLLA-Col micro/nano fiber membrane.
对比例2 PLLA纤维膜的制备Preparation of comparative example 2 PLLA fiber membrane
(1)PLLA静电纺丝液的制备(1) Preparation of PLLA electrospinning solution
首先，将预先称重好的0.5g PLLA固体颗粒加入到4g DCM溶液中，在室温下用磁力搅拌器进行搅拌溶解，直至PLLA溶解形成略粘稠均一的溶液。随后，将2g DMF溶液加入其中，用磁力搅拌器继续搅拌，即可制备出透明均一的PLLA纺丝溶液。First, add 0.5 g of PLLA solid particles weighed in advance to 4 g of DCM solution, stir and dissolve with a magnetic stirrer at room temperature until PLLA dissolves to form a slightly viscous and uniform solution. Subsequently, 2 g of DMF solution was added thereto, and the stirring was continued with a magnetic stirrer to prepare a transparent and uniform PLLA spinning solution.
(2)PLLA静电纺丝纤维膜的制备(2) Preparation of PLLA electrospun fiber membrane
将制备好的PLLA电纺丝溶液加入到连有钝头钢针的10ml的注射器中。注射针的内径为9mm。将一个金属夹子夹在针的头端处，金属夹连接到直流高压电源供应器上。将铝箔附在自制圆形滚筒式上并连接负极来收集电纺丝纤维，精密推进泵被用来控制注射速率。开始纺丝，针头距收集器约15cm，推注速率为60ul/min，电压约15-20kV，最终得到PLLA纤维膜。The prepared PLLA electrospinning solution was added into a 10ml syringe connected with a blunt steel needle. The inner diameter of the injection needle is 9mm. Clip a metal clip over the tip of the needle and connect the metal clip to the DC high voltage power supply. Aluminum foil was attached to a homemade circular drum and connected to the negative electrode to collect the electrospun fibers, and a precision propulsion pump was used to control the injection rate. Start spinning, the needle is about 15cm away from the collector, the injection rate is 60ul/min, the voltage is about 15-20kV, and finally the PLLA fiber membrane is obtained.
对比例3 MS纤维膜的制备Preparation of comparative example 3 MS fiber membrane
(1)微溶胶(MS)纺丝溶液的制备(1) Preparation of microsol (MS) spinning solution
将12mg的透明质酸钠粉末溶解于988mg去离子水中，制备浓度为1.2wt％的HA水溶液。将预先分装好的10ul(10mg)VEGF水溶液(100ug/ml)与50mg1.2wt％HA水溶液充分混匀，最终形成HA质量百分含量为1％wt的HA-VEGF的水溶液60mg。然后，在4g二氯甲烷(DCM)溶液中加入0.01gSpan-80，搅拌均匀后，再上述配置完成的1％HA-VEGF水溶液缓慢滴入，高速搅拌约30min，形成均一稳定的，包裹VEGF的HA微溶胶颗粒的油包水微乳(W/O)。在此油包水微乳中加入0.5g PLLA固体颗粒，在室温下用磁力搅拌器进行搅拌溶解，直至PLLA溶解形成略粘稠均一的溶液。随后，将2g二甲基甲酰胺(DMF)溶液加入其中，用磁力搅拌器继续搅拌，获得微溶胶(MS)纺丝溶液。12 mg of sodium hyaluronate powder was dissolved in 988 mg of deionized water to prepare an HA aqueous solution with a concentration of 1.2 wt%. Fully mix 10ul (10mg) VEGF aqueous solution (100ug/ml) pre-packed with 50mg 1.2wt% HA aqueous solution to form 60mg HA-VEGF aqueous solution with HA mass percentage of 1%wt. Then, add 0.01g of Span-80 to 4g of dichloromethane (DCM) solution, stir evenly, then slowly drop in the above-mentioned 1% HA-VEGF aqueous solution, and stir at high speed for about 30min to form a uniform and stable VEGF-wrapped Water-in-oil microemulsion (W/O) of HA microsol particles. Add 0.5 g of PLLA solid particles to the water-in-oil microemulsion, and stir and dissolve with a magnetic stirrer at room temperature until the PLLA dissolves to form a slightly viscous and uniform solution. Subsequently, 2 g of dimethylformamide (DMF) solution was added thereto, and the stirring was continued with a magnetic stirrer to obtain a microsol (MS) spinning solution.
(2)微溶胶静电纺丝纤维膜的制备(2) Preparation of microsol electrospun fiber membrane
将制备好的微溶胶(MS)纺丝溶液加入到连有钝头钢针的10ml的注射器中。注射针的内径为9mm。将一个金属夹子夹在针的头端处，金属夹连接到直流高压电源供应器上。将铝箔附在自制圆形滚筒式上并连接负极来收集电纺丝纤维，精密推进泵被用来控制注射速率。开始纺丝，针头距收集器约15cm，推注速率为60ul/min，电压约15-20kV，最终得到包裹VEGF-HA的PLLA微米级纤维膜，即微溶胶(MS)纤维膜。The prepared microsol (MS) spinning solution was added to a 10ml syringe connected with a blunt steel needle. The inner diameter of the injection needle is 9 mm. Clip a metal clip over the tip of the needle and connect the metal clip to the DC high voltage power supply. Aluminum foil was attached to a homemade circular drum and connected to the negative electrode to collect the electrospun fibers, and a precision propulsion pump was used to control the injection rate. Start spinning, the needle is about 15cm away from the collector, the injection rate is 60ul/min, the voltage is about 15-20kV, and finally the PLLA micron-sized fiber membrane wrapped with VEGF-HA is obtained, that is, the microsol (MS) fiber membrane.
实施例2理化性能测试实验Embodiment 2 physical and chemical performance test experiment
1.扫描电镜(SEM)1. Scanning Electron Microscope (SEM)
将不同组别的纤维膜裁剪成合适大小后，通过导电胶将样品粘贴于SEM样品台上，使用离子溅射镀膜仪完成喷金过程后，用SEM观察纤维膜形态并拍摄图片，加速电压为10kV。通过Image J软件分别随机计数200根PLLA或MS纤维的直径，结果用纤维直径的分布图和平均直径表示。结果见图2-A、2-C、2D和2E。After cutting different groups of fiber membranes into appropriate sizes, the samples were pasted on the SEM sample stage with conductive glue. After the gold spraying process was completed using an ion sputtering coating device, the morphology of the fiber membranes was observed and pictures were taken with the SEM. The accelerating voltage was 10kV. The diameters of 200 PLLA or MS fibers were randomly counted by Image J software, and the results were expressed by the distribution diagram of fiber diameter and the average diameter. The results are shown in Figures 2-A, 2-C, 2D and 2E.
2.透射电镜(TEM)2. Transmission Electron Microscopy (TEM)
在纺丝过程中使用铜网快速收集少量电纺丝纤维，使用透射电镜来观察单根纤维的内部结构。通过Image J软件计算MS纤维内部的HA核芯直径的平均值。结果见图2B。A copper mesh was used to quickly collect a small amount of electrospun fibers during spinning, and a transmission electron microscope was used to observe the internal structure of individual fibers. The average value of the HA core diameter inside the MS fibers was calculated by Image J software. The results are shown in Figure 2B.
3.动态光散射粒度分析仪(DLS)3. Dynamic light scattering particle size analyzer (DLS)
将制备的HA和DCM乳液体系用石英比色皿装载在动态光散射粒度分析仪(DLS)内，测量HA微溶胶颗粒的尺寸。同时将此乳液体系静置两小时，观测其稳定性。结果见图3-A。The prepared HA and DCM emulsion system was loaded in a dynamic light scattering particle size analyzer (DLS) with a quartz cuvette, and the size of the HA microsol particles was measured. At the same time, the emulsion system was left to stand for two hours to observe its stability. The results are shown in Figure 3-A.
4.接触角测量(WCA)4. Contact angle measurement (WCA)
使用水接触角仪(DSA25S)测量纤维膜的表面静态水接触角，评估其亲水/疏水特性。结果见图3-B。The surface static water contact angle of the fiber membrane was measured with a water contact angle meter (DSA25S) to evaluate its hydrophilic/hydrophobic properties. The results are shown in Figure 3-B.
5.流变测试5. Rheological test
将自组装后的胶原置于流变仪中，在服从应变依赖性的条件下，在37℃下扫描。结果见图3-C。The self-assembled collagen was placed in a rheometer and scanned at 37°C under strain-dependent conditions. The results are shown in Figure 3-C.
6.傅里叶变换红外光谱(FTIR)6. Fourier Transform Infrared Spectroscopy (FTIR)
按照重量比例1：100将HA粉末和Collagen-I固体加入溴化钾(BrK)中，均匀研磨，压片机加压成型备用。将不同组别的纤维膜裁剪成合适大小的片状备用。采用ATR-FTIR(Nicolet 6700)扫描上述样品，扫描次数为128次，分辨率为4cm-1，扫描范围为400～4000cm-1，通过所得曲线分析不通组别化学成份的异同。结果见图3-D。Add HA powder and Collagen-I solid into potassium bromide (BrK) according to the weight ratio of 1:100, grind evenly, press and shape into a tablet machine for later use. Cut different groups of fiber membranes into sheets of appropriate size for later use. ATR-FTIR (Nicolet 6700) was used to scan the above samples, the number of scans was 128, the resolution was 4cm-1, and the scanning range was 400-4000cm-1. The similarities and differences of the chemical components of different groups were analyzed through the obtained curves. The results are shown in Figure 3-D.
7.力学拉伸测试7. Mechanical tensile test
在进行力学测试前，使用磨具将纤维膜制成条状(15.0×3.0×0.1mm)备用。将纤维膜固定于力学测量仪的夹具上，以5mm/min的速度进行拉伸。根据所测得的拉伸数据，绘制相应的应力-应变曲线。结果见图3-E。Before the mechanical test, the fiber membrane was made into strips (15.0×3.0×0.1 mm) by using an abrasive tool for future use. Fix the fiber membrane on the fixture of the mechanical measuring instrument, and stretch it at a speed of 5 mm/min. According to the measured tensile data, draw the corresponding stress-strain curve. The results are shown in Figure 3-E.
8.生物活性因子的体外释放8. In vitro release of bioactive factors
为检测VEGF在体外的释放，取10mg(理论上包含20ng的VEGF)的MS纤维膜，放入50ml离心管内，浸泡于10ml含有1％BSA的PBS溶液中。将离心管置于恒温摇床中震荡，温度为37℃，频率为100圈/min。在规定的时间点(0.5，1，2，4，6，8，10，14，18，23，28d)，尽可能取尽释放液并加入新的PBS溶液。通过ELISA法，测量释放液中VEGF的含量，将所得结果绘制成相应的释放曲线。结果见图3F。To detect the release of VEGF in vitro, 10 mg (theoretically containing 20 ng of VEGF) MS fiber membrane was taken, put into a 50 ml centrifuge tube, and soaked in 10 ml of PBS solution containing 1% BSA. The centrifuge tube was shaken in a constant temperature shaker at a temperature of 37°C and a frequency of 100 cycles/min. At the specified time points (0.5, 1, 2, 4, 6, 8, 10, 14, 18, 23, 28d), the release solution was removed as much as possible and new PBS solution was added. Measure the content of VEGF in the release liquid by ELISA method, and draw the obtained results into corresponding release curves. The results are shown in Figure 3F.
由图1～3可知，本发明制备的仿生骨膜材料大体观察可见人工骨膜亲水性良好，湿润后呈白色半透明薄膜，可操作性良好(图1D)。SEM观察PLLA和MS纤维膜表面形态示，电纺纤维均一、光滑、随机排列(图2A)。PLLA和MS的平均纤维直径分别为0.60±0.15um(图2C)和0.61±0.12um(图2D)，两者没有统计学差异(图3E)。胶原自组装后的电纺膜，形成了明显微/纳米纤维结构，大量蜘蛛网样的胶原纤维沉积于电纺纤维上及其孔隙内部。使用ImageJ软件计算胶原纤维的平均直径约为86.94±35.72nm。从TEM的图片中可以看出，MS纤维有别于PLLA纤维，内部有明显的HA核芯，其结构均一、光滑(图2B)。HA核芯的直径约为0.14±0.02um。From Figures 1 to 3, it can be seen that the bionic periosteum material prepared by the present invention has good hydrophilicity, and the artificial periosteum has good hydrophilicity, and becomes a white translucent film after being wet, with good operability (Figure 1D). SEM observation of the surface morphology of PLLA and MS fiber membranes showed that the electrospun fibers were uniform, smooth, and randomly arranged (Fig. 2A). The mean fiber diameters of PLLA and MS were 0.60±0.15um (Fig. 2C) and 0.61±0.12um (Fig. 2D), respectively, and there was no statistical difference between them (Fig. 3E). The electrospun membrane after collagen self-assembly forms an obvious micro/nano fiber structure, and a large number of spider web-like collagen fibers are deposited on the electrospun fibers and inside the pores. The average diameter of collagen fibers was calculated using ImageJ software to be about 86.94±35.72 nm. It can be seen from the TEM images that MS fibers are different from PLLA fibers in that there is an obvious HA core inside, and its structure is uniform and smooth (Fig. 2B). The diameter of the HA core is about 0.14±0.02um.
图3A显示，HA粒子的直径范围为100-1000nm，其平均尺寸为308.1nm，PDI为0.204，这表示了粒子直径比较均一。此外，静置两小时后粒子直径不会发生太大变化，说明其在DCM中也较为稳定。Figure 3A shows that the diameter range of HA particles is 100-1000 nm, the average size is 308.1 nm, and the PDI is 0.204, which indicates that the particle diameter is relatively uniform. In addition, the particle diameter does not change much after standing for two hours, indicating that it is relatively stable in DCM.
图3B显示，电纺PLLA和MS纤维膜的水接触角分别为128.27°±2.34和126.87°±1.97°，两者差异没有统计学意义，表明包裹于PLLA内部的HA对接触角没有太大影响。然而，在胶原自组装后，所有材料的接触角都显著下降了约50°。PLLA-Col和MS-Col纤维膜的接触角分别为78.97°±4.04°和81.87°±3.48°，这充分显示了一型胶原纳米纤维良好的亲水性能。Figure 3B shows that the water contact angles of the electrospun PLLA and MS fiber membranes were 128.27°±2.34 and 126.87°±1.97°, respectively, and the difference was not statistically significant, indicating that the HA wrapped inside the PLLA did not have much effect on the contact angle. However, after collagen self-assembly, the contact angles of all materials decreased significantly by about 50°. The contact angles of PLLA-Col and MS-Col fiber membranes were 78.97°±4.04° and 81.87°±3.48°, respectively, which fully demonstrated the good hydrophilicity of type I collagen nanofibers.
图3C的流变数据显示，I型胶原完成自组装后为类似于凝胶的状态。The rheological data in Figure 3C shows that collagen type I is in a gel-like state after self-assembly.
图3D的红外图谱显示，单纯的PLLA纤维膜在1750cm-1处有特征性的C＝O伸缩振动带。从图中也可以看出，MS纤维膜与PLLA纤维膜的红外光谱相似，没有显示出特征性的HA带，无法在红外图谱上鉴别两者。单纯的Collagen-I红外图谱，在1650cm-1处显示为特征性酰胺I带(C＝O带)和1550cm-1的酰胺II带(N-H带)。此外，胶原的特征性条带在PLLA-Col和MS-Col纤维膜也可以清晰的观测到。The infrared spectrum of Figure 3D shows that the pure PLLA fiber membrane has a characteristic C=O stretching vibration band at 1750 cm-1. It can also be seen from the figure that the infrared spectra of the MS fiber membrane and the PLLA fiber membrane are similar, and there is no characteristic HA band, and the two cannot be identified on the infrared spectrum. The pure Collagen-I infrared spectrum shows the characteristic amide I band (C=O band) at 1650cm-1 and the amide II band (N-H band) at 1550cm-1. In addition, the characteristic bands of collagen can also be clearly observed in the PLLA-Col and MS-Col fibrous membranes.
图3E的力学拉伸测试显示，PLLA、MS、PLLA-Col和MS-Col纤维膜的最大抗拉强度分别为4.50±0.27MPa、3.92±0.26MPa、5.13±0.28MPa和4.74±0.12MPa。结果显示MS纤维膜的力学强度相比于单纯的PLLA纤维膜有轻度的下降，这可能是由于内部HA核芯的影响。另外，由于大量胶原纤维沉积于电纺纤维表面和其孔隙内部，使得具有多重结构的PLLA-Col和MS-Col纤维膜在一定程度上弥补了这一力学强度的下降。The mechanical tensile test in Fig. 3E showed that the maximum tensile strengths of PLLA, MS, PLLA-Col and MS-Col fiber membranes were 4.50±0.27MPa, 3.92±0.26MPa, 5.13±0.28MPa and 4.74±0.12MPa, respectively. The results showed that the mechanical strength of MS fiber membranes was slightly decreased compared with pure PLLA fiber membranes, which may be due to the influence of the inner HA core. In addition, due to the large amount of collagen fibers deposited on the surface of the electrospun fibers and inside their pores, the PLLA-Col and MS-Col fiber membranes with multiple structures compensated for this decrease in mechanical strength to a certain extent.
图3F的VEGF释放曲线显示，在最初的两天VEGF有一个早期的突释，约36.8％±3.1％的细胞因子被释放，随后释放速度逐渐减慢，至第14天总释放量为68.2％±2.6％，最终释放时间达到28天，总的药物释放量超过了最初药物装载量的80％。The VEGF release curve in Figure 3F shows that there was an early burst release of VEGF in the first two days, about 36.8%±3.1% of the cytokines were released, and then the release rate gradually slowed down to a total release of 68.2% on the 14th day ±2.6%, the final release time reached 28 days, and the total drug release exceeded 80% of the initial drug loading.
综上所述，仿生骨膜具有明显的微/纳米纤维结构，纤维光滑，均一，亲水性良好，为细胞的生存提供了合适微环境，同时具有良好的力学强度，可持续缓慢释放VEGF。In summary, the biomimetic periosteum has obvious micro/nano fiber structure, smooth, uniform, and good hydrophilicity, which provides a suitable microenvironment for the survival of cells, and has good mechanical strength, which can sustainably release VEGF slowly.
实施例3对间充质细胞和血管内皮细胞生长分化的影响Effect of Example 3 on the Growth and Differentiation of Mesenchymal Cells and Vascular Endothelial Cells
1、BMSCs细胞培养1. BMSCs cell culture
在进行细胞培养前，将14mm的普通圆形玻片粘贴于滚筒状的铝箔电纺丝收集装置上，进行电纺丝，制备用于细胞培养的纺丝爬片。将制备好的纺丝爬片置75％的酒精灭菌30min，冲洗3遍完全除去残余酒精后，置于24孔培养板内，按之前的步骤进行胶原自组装，培养基浸泡过夜备用。将大鼠骨髓间充质干细胞(BMSCs)从培养板上消化下来，使用细胞计数器计数3次得到平均细胞浓度。根据特定的实验需要分别在不同组别的纤维膜上接种适宜浓度的细胞，将细胞培养板置于培养箱内，参数为37℃，95％相对湿度和5％CO2分压。每两天更换一次培养液。Before cell culture, a 14mm ordinary circular glass slide was pasted on a drum-shaped aluminum foil electrospinning collection device for electrospinning to prepare a spinning slide for cell culture. The prepared spun slides were sterilized in 75% alcohol for 30 minutes, washed 3 times to completely remove residual alcohol, placed in a 24-well culture plate, and collagen self-assembly was carried out according to the previous steps, and the culture medium was soaked overnight for later use. Rat bone marrow mesenchymal stem cells (BMSCs) were digested from the culture plate, and the average cell concentration was obtained by counting 3 times with a cell counter. According to the needs of specific experiments, cells of appropriate concentration were inoculated on different groups of fiber membranes, and the cell culture plate was placed in an incubator with parameters of 37°C, 95% relative humidity and 5% CO2 partial pressure. The culture medium was changed every two days.
实验分组：(1)普通细胞培养板，未放置材料为Control组；(2)对比例2的PLLA电纺膜，为PLLA组；(3)对比例3的微溶胶纤维膜，添加VEGF，为MS组；(4)对比例1的PLLA-Col纤维膜：PLLA电纺膜+一型胶原自组装，为PLLA-Col组；(5)实施例1的MS-Col纤维膜：微溶胶电纺膜+一型胶原自组装+VEGF，MS-Col组。Experimental grouping: (1) ordinary cell culture plate, no material was placed as Control group; (2) PLLA electrospun membrane of comparative example 2, PLLA group; (3) microsol fiber membrane of comparative example 3, added VEGF, as MS group; (4) PLLA-Col fiber membrane of Comparative Example 1: PLLA electrospun membrane+type 1 collagen self-assembly, which is PLLA-Col group; (5) MS-Col fiber membrane of Example 1: microsol electrospinning Membrane + type 1 collagen self-assembly + VEGF, MS-Col group.
2.细胞活/死荧光染色检测2. Cell live/dead fluorescent staining detection
通过细胞的活/死荧光染色，评估细胞在不同材料上的活力。按照之前的方法，以4×104细胞/ml的浓度在24孔板内接种细胞，使用配置好的活/死细胞工作染液在室温条件下进行30分钟细胞染色。在荧光显微镜下观察，活细胞被绿色荧光染色，死细胞被红色荧光染色，结果见图4。Assess cell viability on different materials by live/dead fluorescent staining of cells. According to the previous method, cells were seeded in a 24-well plate at a concentration of 4×104 cells/ml, and the cells were stained for 30 minutes at room temperature using the prepared live/dead cell working dye solution. Observed under a fluorescent microscope, live cells were stained with green fluorescence, and dead cells were stained with red fluorescence. The results are shown in Figure 4.
3.CCK-8细胞增殖实验3. CCK-8 cell proliferation assay
按照上述培养方法在24孔培养板内分别接种1×105细胞，将未加材料的普通培养板设为对照组，以对比材料和培养板之间的差异。将CCK-8试剂与培养基以1：9的比例进行混合，在不同的时间点(1、3、5、7天)取代培养基，于培养箱内培养4小时。将100ul的混合培养液从24孔板内转移到96孔板内，使用酶标仪检测450nm处的吸光度，统计细胞培养1、3、5、7天细胞增殖情况，结果见图5。According to the above culture method, 1×105 cells were inoculated in 24-well culture plates respectively, and the common culture plate without materials was set as the control group to compare the difference between the materials and the culture plates. The CCK-8 reagent was mixed with the medium at a ratio of 1:9, and the medium was replaced at different time points (1, 3, 5, 7 days), and cultured in an incubator for 4 hours. Transfer 100ul of the mixed culture solution from the 24-well plate to the 96-well plate, use a microplate reader to detect the absorbance at 450nm, and count the cell proliferation on the 1st, 3rd, 5th, and 7th days of cell culture. The results are shown in Figure 5.
4.细胞黏附形态观察4. Observation of cell adhesion morphology
为了评估细胞在不同电纺纤维膜上的铺展形态，将4×104的细胞接种于置入24孔培养板内的纤维膜表面，放入培养箱内培养24小时。将细胞培养板取出，吸进培养液，使用4％的多聚甲醛固定细胞15分钟，冲洗3遍去除残余的多聚甲醛。随后，用0.3％的Triton X-100进行细胞膜打孔，时间10分钟，冲洗三遍。最后，在避光条件下使用鬼笔环肽和DAPI进行细胞骨架和细胞核染色，时间分别为30分钟。结果见图6。In order to evaluate the spreading morphology of cells on different electrospun fiber membranes, 4×104 cells were seeded on the surface of fiber membranes placed in 24-well culture plates, and cultured in an incubator for 24 hours. The cell culture plate was taken out, the culture medium was aspirated, the cells were fixed with 4% paraformaldehyde for 15 minutes, and the residual paraformaldehyde was removed by washing 3 times. Subsequently, the cell membrane was perforated with 0.3% Triton X-100 for 10 minutes and washed three times. Finally, cytoskeleton and nuclei were stained using phalloidin and DAPI for 30 min, respectively, in the dark. The results are shown in Figure 6.
5.细胞黏附机制研究5. Research on cell adhesion mechanism
为了确认微/纳米纤维结构改变细胞黏附形态的机制，使用Integrin和Vinculin抗体分别对细胞进行免疫荧光染色。在不同纤维膜上培养后，使用上述相同的方法对BMSCs进行固定，打孔，冲洗，然后使用5％的BSA溶液4℃过夜封闭。再次进行PBS冲洗后，使用Integrinβ1或vinculin一抗进行4℃过夜孵育，然后加入相应的二抗室温孵育。最后分别进行细胞骨架和细胞核的染色。将细胞爬片从培养板中取出，放置在载玻片上，于荧光显微镜下进行观察拍摄。使用Image J软件对荧光强度进行半定量分析。结果见图7和图8。To confirm the mechanism by which the micro/nanofibrous structure changes the morphology of cell adhesion, the cells were immunofluorescently stained using Integrin and Vinculin antibodies, respectively. After being cultured on different fiber membranes, BMSCs were fixed using the same method as above, punched, washed, and then blocked overnight at 4°C with 5% BSA solution. After washing with PBS again, use the Integrinβ1 or vinculin primary antibody to incubate overnight at 4°C, and then add the corresponding secondary antibody to incubate at room temperature. Finally, the cytoskeleton and nucleus were stained separately. The cell slides were taken out from the culture plate, placed on a glass slide, and observed and photographed under a fluorescent microscope. The fluorescence intensity was semi-quantitatively analyzed using Image J software. The results are shown in Figures 7 and 8.
6.细胞成骨分化检测6. Osteogenic Differentiation Detection of Cells
以相同的方法制备好载有特定纤维膜的细胞培养板备用，将普通培养板包被0.1％明胶作为Control组。将2×104个细胞/孔接种于纤维膜或培养板上，先以完全培养基进行培养，当细胞融合度达到60％时，去除完全培养基，使用大鼠成骨诱导培养基进行分化诱导，定时观察细胞生长情况，每2-3天换液一次。在诱导7天后，使用ALP染色试剂盒和ALP活性定量试剂盒分别进行检测，结果用显微镜照片和活性定量柱状图表示，结果见图9。将细胞诱导21天后，使用茜素红染色试剂对钙结节进行染色，显微镜拍取照片后，使用高氯酸溶解钙结节，在420nm处检测吸光度，定量结果用柱状图进行表示，，结果见图10。Cell culture plates loaded with specific fiber membranes were prepared in the same way for later use, and common culture plates were coated with 0.1% gelatin as the Control group. Inoculate 2×104 cells/well on fiber membrane or culture plate, culture with complete medium first, remove complete medium when cell confluency reaches 60%, and use rat osteogenic induction medium for differentiation induction , regularly observe the cell growth, and change the medium every 2-3 days. After 7 days of induction, the ALP staining kit and the ALP activity quantification kit were used for detection respectively, and the results were shown in micrographs and activity quantification histograms, and the results are shown in Figure 9. After the cells were induced for 21 days, the calcium nodules were stained with alizarin red staining reagent. After taking pictures under the microscope, the calcium nodules were dissolved with perchloric acid, and the absorbance was detected at 420nm. The quantitative results were expressed in histograms. See Figure 10.
7.体外血管内皮细胞成管试验7. Tube formation test of vascular endothelial cells in vitro
在进行人脐静脉血管内皮细胞(HUVECs)成管实验前，在24孔培养板内，每孔加入100μL的生长因子减少型基质胶(Corning,美国)，将培养板置入37℃培养箱内使液态的基质胶成胶备用。将MS纤维膜和PLLA纤维膜提前分别置于Trans-well板(Corning,美国)的上层(0.4μm)小室内过夜浸泡后，在下层小室内以5×104细胞/ml的浓度接种HUVECs。将培养板置于37℃，5％CO2的条件下进行培养促使内皮细胞成管。分别于3小时和6小时后，吸除培养基，以4％多聚甲醛溶液进行固定之后，冲洗3遍。采用上述同样的方法对HUVECs的细胞骨架和细胞核进行染色。使用荧光显微镜进行观察和图片拍摄。使用Image J软件对拍摄后的图片进行处理计算，得到平均的血管形成参数。结果见图11。Before the human umbilical vein endothelial cells (HUVECs) tube formation experiment, 100 μL of growth factor-reduced Matrigel (Corning, USA) was added to each well of the 24-well culture plate, and the culture plate was placed in a 37°C incubator. Make the liquid matrigel into a gel for later use. MS fiber membranes and PLLA fiber membranes were placed in the upper (0.4 μm) chamber of the Trans-well plate (Corning, USA) in advance and soaked overnight, and HUVECs were inoculated at a concentration of 5×104 cells/ml in the lower chamber. The culture plate was cultured at 37°C and 5% CO2 to promote the tube formation of endothelial cells. After 3 hours and 6 hours respectively, the culture medium was sucked off, fixed with 4% paraformaldehyde solution, and washed 3 times. The cytoskeleton and nucleus of HUVECs were stained by the same method as above. Observations and pictures were taken using a fluorescence microscope. Image J software was used to process and calculate the captured pictures to obtain the average angiogenesis parameters. The results are shown in Figure 11.
结果分析：Result analysis:
由图4可知，细胞在不同组别的纤维膜上均可以生长。但是由于纯PLLA纤维膜较差的生物相容性，在其上生长的活细胞数量明显少于PLLA-Col胶原自组装组，并且死细胞数量也较多。同时，通过微溶胶电纺技术添加HA和VEGF制备而成的MS组与纯PLLA组对比没有差异，这可能是由于添加的HA都在纤维内部，对PLLA的疏水性没有改善。最后，相比于Control组，胶原自组装组(PLLA-Col和MS-Col)在细胞活/死染色图片对比上无明显差异。It can be seen from Figure 4 that cells can grow on different groups of fibrous membranes. However, due to the poor biocompatibility of the pure PLLA fibrous membrane, the number of living cells grown on it was significantly less than that of the PLLA-Col collagen self-assembled group, and the number of dead cells was also higher. At the same time, there was no difference between the MS group prepared by adding HA and VEGF by microsol electrospinning technology and the pure PLLA group, which may be because the added HA was inside the fiber and did not improve the hydrophobicity of PLLA. Finally, compared with the Control group, the collagen self-assembly group (PLLA-Col and MS-Col) had no significant difference in the comparison of cell live/dead staining pictures.
由图5可知，BMSCs能在各材料表面进行增殖生长，从1至7天各组细胞数量总体呈上升趋势，Control组的细胞增殖速率优于各材料组，其余组差异的对比结果与之前的细胞活性检测实验一致。总的来说，虽然由于普通培养板专业的细胞培养性能，材料组的细胞增殖速率均低于Control组，但是具有微/纳米纤维结构的纤维膜相比于单纯的高分子聚合物纤维膜，生物相容性已经有了显著提高。It can be seen from Figure 5 that BMSCs can proliferate and grow on the surface of each material, and the number of cells in each group shows an overall upward trend from 1 to 7 days. The cell proliferation rate of the Control group is better than that of each material group. The cell viability detection experiment was consistent. In general, although the cell proliferation rate of the material group was lower than that of the Control group due to the professional cell culture performance of the ordinary culture plate, the fiber membrane with micro/nano fiber structure was compared with the pure polymer fiber membrane. Biocompatibility has been significantly improved.
由图6可知，所有组别的纤维膜上均有一定数量的细胞黏附生长，但是PLLA和MS组别上细胞数量较少，细胞形态瘦小、细长，伪足少。而胶原-PLLA网络状结构对细胞的黏附有良好的促进作用，细胞铺展良好。通过细胞骨架图片对细胞的铺展面积进行定量显示，通过胶原改性的电纺纤维膜其铺展面积(6453±1479μm2)约是单纯PLLA或MS组的4.5倍。It can be seen from Figure 6 that a certain number of cells adhered to and grew on the fibrous membranes of all groups, but the number of cells in the PLLA and MS groups was small, the cells were thin, elongated, and had few pseudopodia. The collagen-PLLA network structure has a good promotion effect on cell adhesion, and the cells spread well. Quantification of cell spreading area by cytoskeleton pictures showed that the spreading area (6453±1479μm2) of collagen-modified electrospun fiber membrane was about 4.5 times that of pure PLLA or MS group.
Integrin是一种跨膜蛋白受体，它通过调节细胞外基质与细胞骨架间的链接，在信号转导过程起着重要作用。从图7中可以看出，相比于单纯的PLLA或者MS纤维膜，培养在PLLA-Col或MS-Col纤维膜上的细胞表达了更高红色荧光信号。这代表了胶原自组装组能显著促进integrinβ1亚单位的表达。而PLLA组和MS组两者没有显著差异。Integrin is a transmembrane protein receptor that plays an important role in signal transduction by regulating the link between the extracellular matrix and the cytoskeleton. It can be seen from Figure 7 that compared with pure PLLA or MS fiber membranes, cells cultured on PLLA-Col or MS-Col fiber membranes expressed higher red fluorescence signals. This represents that the collagen self-assembly group can significantly promote the expression of integrinβ1 subunit. There was no significant difference between the PLLA group and the MS group.
Vinculin是黏着斑(FA)的主要组成蛋白，integrin可以促进vinculin的表达。Vinculin可以加强细胞-细胞、细胞-外基质的黏附，以此介导机械信号对细胞行为的影响。图8显示，PLLA-Col和MS-Col组表达的vinculin在胞质内连接于肌动蛋白纤维上，并且可以观察到被募集到黏着斑复合体的vinculin聚集在细胞边缘的丝状或板状伪足纤维束的终末端，形成了致密的点状红色荧光。而PLLA组和MS组仅仅显示了少量的vinculin表达，并且无点状浓聚形成。Vinculin is the main component protein of focal adhesion (FA), and integrin can promote the expression of vinculin. Vinculin can strengthen cell-cell and cell-extramatrix adhesion, thereby mediating the influence of mechanical signals on cell behavior. Figure 8 shows that the vinculin expressed in the PLLA-Col and MS-Col groups is attached to the actin fibers in the cytoplasm, and it can be observed that the vinculin recruited to the focal adhesion complex gathers in filaments or plates at the cell edge The terminal ends of the pseudopodia fiber bundles formed dense dot-like red fluorescence. However, PLLA group and MS group only showed a small amount of vinculin expression, and no punctate condensate was formed.
综上所述，一型胶原纤维沉积于电纺纤维膜上构成的微/纳米纤维结构极大了改变了细胞的黏附模式。In summary, the micro/nanofibrous structure formed by the deposition of type 1 collagen fibers on the electrospun fibrous membrane greatly changed the adhesion mode of cells.
由图9～10可知，相比PLLA组和MS组，胶原自组装组的ALP染色和茜素红染色均最深，表明该组骨向分化程度最高。It can be seen from Figures 9-10 that, compared with the PLLA group and the MS group, the ALP staining and Alizarin red staining in the collagen self-assembly group were the deepest, indicating that this group had the highest degree of osteogenic differentiation.
由图11可知，可以释放VEGF的MS组在培养3h和6h后，从内皮细胞聚集处分支出来的管状结构构成了大量原始的血管样网状结构，而PLLA组仅仅有少量不明显的管状结构可以观察到。使用Image J软件对管状节点数量和管状结构长度进行计数，MS组在3h和6h的平均管状长度分别为4672±191μm和6653±768μm，而对于没有VEGF的PLLA组数据分别为2993±460μm和4247±250μm，两者有显著差异。平均节点数量的计数趋势与管状长度的趋势一致。It can be seen from Figure 11 that after 3 hours and 6 hours of culture in the MS group that can release VEGF, the tubular structures branched from the endothelial cell aggregation formed a large number of original blood vessel-like network structures, while the PLLA group only had a small amount of inconspicuous tubular structures It can be observed. Using Image J software to count the number of tubular nodes and the length of tubular structures, the average tubular lengths of the MS group at 3h and 6h were 4672±191μm and 6653±768μm, respectively, while the data of the PLLA group without VEGF were 2993±460μm and 4247 ±250μm, there is a significant difference between the two. The count trend for the mean number of nodes is consistent with the trend for tubular length.
以上结果显示，本发明提供的仿生骨膜具有良好的生物相容性，可促进间充质细胞的黏附、增殖以及成骨分化从而构成生发层，并且体外释放的VEGF具有促进血管化的能力。The above results show that the bionic periosteum provided by the present invention has good biocompatibility, can promote the adhesion, proliferation and osteogenic differentiation of mesenchymal cells to form the germinal layer, and the VEGF released in vitro has the ability to promote vascularization.
实施例4对骨膜与骨缺损修复的体内评价Example 4 In vivo evaluation of periosteum and bone defect repair
1、大鼠颅骨骨膜与骨缺损模型的构建1. Construction of rat skull periosteum and bone defect model
取30只SD大鼠随机分为5组。即未植入材料的Control组，植入对比例2PLLA纤维膜的PLLA组，植入对比例3MS纤维膜的MS组，植入对比例1PLLA-Col纤维膜的PLLA-Col组，植入实施例1MS-Col纤维膜的MS-Col组。实验评价指标为观察手术4周和8周后骨膜以及骨缺损修复的情况。30 SD rats were randomly divided into 5 groups. That is, the Control group without material implantation, the PLLA group implanted with comparative example 2 PLLA fiber membrane, the MS group implanted with comparative example 3MS fiber membrane, the PLLA-Col group implanted with comparative example 1 PLLA-Col fiber membrane, and the implanted embodiment 1 MS-Col set of MS-Col fiber membranes. The experimental evaluation index is to observe the repair of periosteum and bone defect after 4 and 8 weeks of operation.
采用2％的戊巴比妥钠溶液腹腔注射对SD大鼠进行麻醉，注射剂量为2.5ml/kg。充分麻醉后，剔除大鼠颅顶部的毛发，安尔碘消毒，沿颅骨纵轴做一正中的长切口，小心逐层分离皮下组织直至暴露颅骨。剥离大鼠颅骨膜后，使用牙科钻在颅骨矢状缝两侧分别钻取一个5mm的圆形骨缺损区域，最大限度模拟骨缺损以及骨膜缺损的条件。根据分组，将骨缺损区域覆盖不同的纤维膜，小心缝合切口，再次消毒。每天肌注青霉素，持续3天。The SD rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium solution, and the injection dose was 2.5 ml/kg. After adequate anesthesia, the hair on the top of the skull of the rat was shaved off, disinfected with aner iodine, a long central incision was made along the longitudinal axis of the skull, and the subcutaneous tissue was carefully separated layer by layer until the skull was exposed. After peeling off the rat cranial periosteum, a 5mm circular bone defect area was drilled on both sides of the sagittal suture of the skull using a dental drill to maximize the simulation of bone defect and periosteal defect conditions. According to the grouping, the bone defect area was covered with different fibrous membranes, the incision was carefully sutured, and disinfected again. Daily intramuscular injection of penicillin for 3 days.
2、Micro-CT分析2. Micro-CT analysis
在手术后4周和8周，对SD大鼠实施安乐死，处死后收取大鼠的颅骨标本，并用10％福尔马林溶液固定备用。首先使用Micro-CT评估大鼠颅骨缺损区域的修复情况，参数设置如下：65kV、385mA。使用Mimic软件对扫描的颅骨图片进行3D重建。使用CT Analyzer软件指定合适的圆柱形区域为感兴趣区域(ROI)，分析其骨组织和总的组织体积比得出BV/TV(骨组织体积/总体积)。结果见图12。At 4 weeks and 8 weeks after the operation, the SD rats were euthanized, and the skull specimens of the rats were collected and fixed with 10% formalin solution for later use. First, Micro-CT was used to evaluate the repair of the rat skull defect area, and the parameters were set as follows: 65kV, 385mA. The scanned skull images were reconstructed in 3D using Mimic software. Use CT Analyzer software to designate a suitable cylindrical area as a region of interest (ROI), and analyze its bone tissue and total tissue volume ratio to obtain BV/TV (bone tissue volume/total volume). The results are shown in Figure 12.
3、组织学分析3. Histological analysis
将收取的标本进行固定后，使用10％的甲酸溶液于室温下脱钙1周。随后，使用乙醇溶液进行梯度脱水，然后石蜡包埋。使用切片机，通过骨缺损中心，将包埋好的标本切片，厚度为5um。将组织切片进行CD31和Periostin(骨膜蛋白)的免疫组化染色，分别评估血管再生和骨膜的修复情况。结果见图13～14。After the collected specimens were fixed, they were decalcified with 10% formic acid solution at room temperature for 1 week. Subsequently, gradient dehydration was performed using ethanol solution, followed by paraffin embedding. Using a microtome, slice the embedded specimen through the center of the bone defect with a thickness of 5um. The tissue sections were immunohistochemically stained for CD31 and Periostin (periostin) to evaluate angiogenesis and periosteum repair, respectively. The results are shown in Figures 13-14.
结果分析：Result analysis:
在术后4周和8周时，micro-CT被用来进一步从宏观角度分析新骨修复的情况。由图12可以看出，MS-Col组与其他组别之间有着显著差异。MS-Col组的骨愈合情况最佳，显示了微/纳米结构和VEGF对骨修复的协同作用，而Control组仅仅形成了非常少量的骨组织。PLLA组的表现略好于Control组，这可能是由于其在缺损表面起到了一个临时的物理屏障作用，隔绝了外周软组织与骨髓腔的内环境，减少了外周软组织的入侵和骨髓间充质细胞的外溢。添加VEGF的MS组增强了再血管化能力，使其在成骨表现上优于单纯的PLLA组。此外，MS-Col组与普通的PLLA电纺纤维膜相比，两者成骨的模式有显著差异。PLLA组的骨组织倾向于从缺损的边缘向中心生长。而胶原自组装组的成骨模式除上述特点外，还表现为在不与骨缺损边缘连接的中心部位进行成骨，这代表了在纤维膜内面的直接骨化。结果表明，本发明提供的外源性的仿生骨膜能够模拟骨膜的发育过程，诱导生发层的形成和进行膜内成骨。最后，对micro-CT数据进行BV/TV分析，其结果趋势与上述描述一致。At 4 and 8 weeks postoperatively, micro-CT was used to further macroscopically analyze new bone repair. It can be seen from Figure 12 that there are significant differences between the MS-Col group and other groups. The MS-Col group had the best bone healing, showing the synergistic effect of micro/nanostructures and VEGF on bone repair, while the Control group only formed a very small amount of bone tissue. The performance of the PLLA group was slightly better than that of the Control group, which may be due to its role as a temporary physical barrier on the defect surface, isolating the internal environment of the peripheral soft tissue and the bone marrow cavity, reducing the invasion of peripheral soft tissue and bone marrow mesenchymal cells spillover. The addition of VEGF in the MS group enhanced revascularization, making it superior to the PLLA-only group in terms of osteogenesis. In addition, there was a significant difference in the osteogenesis pattern between the MS-Col group and the common PLLA electrospun fiber membrane. The bone tissue in the PLLA group tended to grow from the edge of the defect to the center. In addition to the above-mentioned characteristics, the osteogenesis mode of the collagen self-assembled group also showed that the osteogenesis occurred in the central part that was not connected with the edge of the bone defect, which represented the direct ossification on the inner surface of the fibrous membrane. The results show that the exogenous bionic periosteum provided by the invention can simulate the development process of the periosteum, induce the formation of the germinal layer and carry out intramembranous osteogenesis. Finally, BV/TV analysis was performed on the micro-CT data, and the trend of the results was consistent with the above description.
由图13可知，血小板内皮细胞黏附分子(PECAM-1)，也被称作CD31，广泛表达于内皮细胞上，可以介导细胞间的黏附。因此，CD31被广泛的用于评估血管再生的状况。在图13中，骨缺损区域的新生血管中的CD31被抗体标记，表现为棕色、圆形或椭圆形的结构(图中用黄色箭头指出)。与体外内皮细胞成管实验的结果相同，在4周时，MS-Col组表现出更为强大的早期促血管生成能力，新生骨组织周围有大量血管，效果明显优于其他各组。It can be known from FIG. 13 that platelet endothelial cell adhesion molecule (PECAM-1), also known as CD31, is widely expressed on endothelial cells and can mediate intercellular adhesion. Therefore, CD31 is widely used to assess the status of angiogenesis. In FIG. 13 , CD31 in the new blood vessels in the bone defect area was labeled by the antibody, showing brown, round or oval structures (indicated by yellow arrows in the figure). Similar to the results of endothelial cell tube formation experiments in vitro, at 4 weeks, the MS-Col group showed a stronger ability to promote early angiogenesis, and there were a large number of blood vessels around the new bone tissue, which was significantly better than other groups.
图14中，骨膜蛋白(Periostin)又被称为成骨细胞特异性因子2(OSF-2)，由骨膜成骨细胞和前成骨细胞表达，存在于受应力刺激的结缔组织内，尤其是骨膜组织。由于不存在于骨基质中，所以骨膜蛋白可以被用作骨膜特异性标记物。从图14的免疫组化分析中可以看出，在MS-Col组，再生的骨组织表面形成了一层高表达骨膜蛋白的组织，并且呈线性排列(图中用黑色箭头指出)。这一现象从分子学角度印证了骨膜的再生。相比于MS-Col组，其他的组别仅仅只有少量的骨膜蛋白以分散、无序的形式分布于细胞外基质中，无特殊的结构。In Figure 14, periostin (Periostin), also known as osteoblast-specific factor 2 (OSF-2), is expressed by periosteal osteoblasts and pre-osteoblasts, and exists in connective tissue stimulated by stress, especially Periosteal tissue. Since it is not present in the bone matrix, periostin can be used as a periosteum-specific marker. From the immunohistochemical analysis in Figure 14, it can be seen that in the MS-Col group, a layer of tissue highly expressing periostin was formed on the surface of the regenerated bone tissue, and it was arranged linearly (indicated by black arrows in the figure). This phenomenon confirms the regeneration of periosteum from molecular point of view. Compared with the MS-Col group, only a small amount of periostin in the other groups was distributed in the extracellular matrix in a dispersed and disordered form, without special structure.
综上所述，本发明提供的微/纳米仿生骨膜材料具有显著地促进骨膜与骨再生的能力，效果明显优于其他各组。To sum up, the micro/nano bionic periosteum material provided by the present invention has the ability to significantly promote the regeneration of periosteum and bone, and the effect is obviously better than that of other groups.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.
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| CN201910578078.4ACN110201226B (en) | 2019-06-28 | 2019-06-28 | A kind of micro/nano bionic periosteal material and preparation method thereof |
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