技术领域Technical Field
本发明涉及骨修复技术领域，具体而言，涉及一种基于形状记忆材料的骨组织支架及其制备方法。The present invention relates to the technical field of bone repair, and in particular to a bone tissue scaffold based on shape memory material and a preparation method thereof.
背景技术Background technique
创伤、手术或肿瘤引起的骨缺损是临床常见疾病，目前骨组织支架的微创植入和功能重建能力有限，影响了这些方法的临床应用。常用的治疗方法为自体组织移植修复、同种异体移植组织替代或器官移植。然而自体组织移植往往伴有新的创伤或受供体部位条件限制，难以达到治疗效果。异体组织移植存在供体来源和免疫排斥的问题。特别是对于较大的骨缺损，传统的治疗方法已不能满足医疗需求。此外，人体在太空会受到微重力、低温、辐射等多种极端环境的影响。其中，由微重力环境所导致的骨愈合缓慢、骨不连及骨流失等问题是现阶段关乎深空探索的战略瓶颈问题。然而在骨再生领域，传统支架在力学上仅起到局部支撑作用，尚无法补偿微重力下的周期性应力需求，对促进骨修复帮助不大。Bone defects caused by trauma, surgery or tumors are common clinical diseases. The current minimally invasive implantation and functional reconstruction capabilities of bone tissue scaffolds are limited, which affects the clinical application of these methods. Common treatment methods are autologous tissue transplantation repair, allogeneic transplantation tissue replacement or organ transplantation. However, autologous tissue transplantation is often accompanied by new trauma or is limited by the conditions of the donor site, making it difficult to achieve therapeutic effects. Allogeneic tissue transplantation has problems with donor sources and immune rejection. Especially for larger bone defects, traditional treatment methods can no longer meet medical needs. In addition, the human body will be affected by a variety of extreme environments such as microgravity, low temperature, and radiation in space. Among them, problems such as slow bone healing, bone nonunion and bone loss caused by the microgravity environment are strategic bottlenecks related to deep space exploration at this stage. However, in the field of bone regeneration, traditional scaffolds only play a local supporting role in mechanics, and cannot compensate for the periodic stress requirements under microgravity, which is not very helpful in promoting bone repair.
发明内容Summary of the invention
本发明所要解决的问题是制备一种骨组织支架，不仅起到基本的支撑作用，还能够补偿微重力环境下的周期性应力需求，提高成骨效率。The problem to be solved by the present invention is to prepare a bone tissue scaffold that not only plays a basic supporting role, but also can compensate for the periodic stress requirements in a microgravity environment and improve the bone formation efficiency.
为解决上述问题，本发明提供一种基于形状记忆材料的骨组织支架的制备方法，包括：In order to solve the above problems, the present invention provides a method for preparing a bone tissue scaffold based on a shape memory material, comprising:
以形状记忆聚合物、纳米颗粒、压电材料、Mxenes为原料制备形状记忆打印线材；Shape memory printing filaments are prepared using shape memory polymers, nanoparticles, piezoelectric materials, and Mxenes as raw materials;
根据骨组织材料的形态、结构，利用控制方程进行骨组织支架的仿生设计，得到骨组织支架的模型；According to the morphology and structure of bone tissue materials, the control equation is used to carry out the bionic design of bone tissue scaffolds and obtain the model of bone tissue scaffolds;
以所述形状记忆打印线材为原料，根据所述骨组织支架的模型，利用4D打印方法制备基于形状记忆的骨组织支架。The shape memory printing wire is used as a raw material, and according to the model of the bone tissue scaffold, a shape memory-based bone tissue scaffold is prepared by a 4D printing method.
较佳地，所述压电材料为钛酸钡、氧化锌纳米颗粒、铌酸钾钠和聚偏氟乙烯纳米材料中的一种或多种。Preferably, the piezoelectric material is one or more of barium titanate, zinc oxide nanoparticles, potassium sodium niobate and polyvinylidene fluoride nanomaterials.
较佳地，所述压电材料的体积分数为1％-20％。Preferably, the volume fraction of the piezoelectric material is 1%-20%.
较佳地，所述纳米颗粒包括Fe3O4纳米颗粒、纳米金颗粒、碳粉、炭黑、石墨烯、碳纳米管和镍粉中的一种或多种。Preferably, the nanoparticles include one or more of Fe3 O4 nanoparticles, nanogold particles, carbon powder, carbon black, graphene, carbon nanotubes and nickel powder.
较佳地，所述纳米颗粒的体积分数为1％-20％。Preferably, the volume fraction of the nanoparticles is 1%-20%.
较佳地，所述Mxenes的体积分数≤1％。Preferably, the volume fraction of the Mxenes is ≤1%.
较佳地，所述形状记忆聚合物包括形状记忆聚氨酯、形状记忆聚乳酸、形状记忆聚己内酯、形状记忆壳聚糖及其复合物和衍生物中的一种或多种。Preferably, the shape memory polymer includes one or more of shape memory polyurethane, shape memory polylactic acid, shape memory polycaprolactone, shape memory chitosan, and complexes and derivatives thereof.
较佳地，所述根据骨组织材料的形态、结构，利用控制方程进行骨组织支架的仿生设计，得到骨组织支架的模型，包括：Preferably, the bionic design of the bone tissue scaffold is performed using a control equation according to the morphology and structure of the bone tissue material to obtain a model of the bone tissue scaffold, including:
利用计算机断层扫描获得骨小梁的模型，经过高斯滤波和阈值处理得到矿化骨相的三维二值图像；The trabecular bone model was obtained by computed tomography, and the three-dimensional binary image of the mineralized bone phase was obtained after Gaussian filtering and threshold processing;
对所述三维二值图像利用移动立方体算法重构骨小梁表面的三角网格，并采用隐式曲面光顺对所述三角网格进行平滑处理；Reconstructing the triangular mesh of the trabecular surface using the marching cube algorithm for the three-dimensional binary image, and smoothing the triangular mesh using implicit surface smoothing;
利用多尺度曲面拟合算法，离散所述三角形网格的顶点，估算出骨小梁表面的主曲率，将二阶多项式拟合到所述三角网格顶点周围的局部邻域进行曲率估计，并利用Minkowski函数量化骨小梁的局部和全局几何特征；Using a multi-scale surface fitting algorithm, the vertices of the triangular mesh are discretized to estimate the principal curvature of the trabecular surface, a second-order polynomial is fitted to the local neighborhood around the vertices of the triangular mesh to estimate the curvature, and the local and global geometric features of the trabecular bone are quantified using a Minkowski function;
结合极小曲面的几何建模方法，通过控制方程对支架进行仿生设计，得到所述骨组织支架的模型。Combined with the geometric modeling method of minimal surfaces, the scaffold is biomimetically designed through control equations to obtain the model of the bone tissue scaffold.
较佳地，所述控制方程包括下述公式中的任意一个：Preferably, the control equation includes any one of the following formulas:
F1(x,y,z)＝α1cos(x)+β1cos(y)+γ1cos(z)+δ1 (1)；F1 (x, y, z) = α1 cos(x) + β1 cos(y) + γ1 cos(z) + δ1 (1);
F2(x,y,z)＝α2cos(x)sin(y)+β2cos(y)sin(z)+γ2cos(z)sin(x)+δ2 (2)；F2 (x,y,z)＝α2 cos(x)sin(y)+β2 cos(y)sin(z)+γ2 cos(z)sin(x)+δ2 (2);
其中，Fi(x,y,z)表示第i个控制函数，i＝1,2,3，x、y、z分别表示笛卡尔坐标中的三个空间方向，αi表示第i个控制函数中的第一形状控制参数，βi表示第i个控制函数中的第二形状控制参数,γi表示第i个控制函数中的第三形状控制参数，λ表示第四形状控制参数，δi表示第i个控制函数中的控制实体比例的参数。Among them,Fi (x, y, z) represents the i-th control function, i = 1, 2, 3, x, y, z represent the three spatial directions in Cartesian coordinates respectively,αi represents the first shape control parameter in the i-th control function,βi represents the second shape control parameter in the i-th control function,γi represents the third shape control parameter in the i-th control function, λ represents the fourth shape control parameter, andδi represents the parameter of the control entity proportion in the i-th control function.
本发明还提供一种基于形状记忆材料的骨组织支架，采用所述的基于形状记忆材料的骨组织支架的制备方法制备得到。The present invention also provides a bone tissue scaffold based on a shape memory material, which is prepared by using the preparation method of the bone tissue scaffold based on a shape memory material.
本发明通过在用于打印支架的线材中添加压电材料和MXenes(二维过渡金属碳化物和氮化物)，使支架具有压电功能和摩擦电功能，支架压电性与人体骨骼相似，可以通过提供不同形式的电信号来调节细胞行为，同时具有高效的机电转换能力，运动过程中产生的脉冲直流信号不仅可以引导细胞平行于电场的方向，而且对成骨分化也有积极作用。The present invention adds piezoelectric materials and MXenes (two-dimensional transition metal carbides and nitrides) to the wire used to print the stent, so that the stent has piezoelectric and triboelectric functions. The piezoelectricity of the stent is similar to that of human bones, and can regulate cell behavior by providing electrical signals in different forms. At the same time, it has efficient electromechanical conversion capabilities. The pulsed DC signal generated during the movement can not only guide the cells parallel to the direction of the electric field, but also has a positive effect on osteogenic differentiation.
本发明通过对骨组织内部多层级微结构的仿生设计，改善支架的生物相容性，促进与周围骨组织的骨整合，且支架兼具形状记忆、可降解等功能，可通过微创手术以收缩的状态植入到骨缺损部位，并在一定的刺激下回复到其初始形状，并与骨缺损边界接触良好。利用其双向形状记忆效应在一定的刺激条件下完成非接触式往复形变，具有形变可逆、频率可控、条件温和耐疲劳等特性，在微重力条件下提供成骨所需的周期性应力补偿，进而对细胞产生局部机械应力，还可进一步调节细胞行为，导致细胞交互促进成骨，加速骨缺损在微重力条件下的修复过程，实现在微重力条件下协同促进成骨。The present invention improves the biocompatibility of the scaffold and promotes bone integration with the surrounding bone tissue through the bionic design of the multi-level microstructure inside the bone tissue. The scaffold has the functions of shape memory and degradability. It can be implanted into the bone defect site in a contracted state through minimally invasive surgery, and return to its initial shape under certain stimulation, and has good contact with the bone defect boundary. The bidirectional shape memory effect is used to complete non-contact reciprocating deformation under certain stimulation conditions. It has the characteristics of reversible deformation, controllable frequency, mild conditions and fatigue resistance. It provides the periodic stress compensation required for osteogenesis under microgravity conditions, thereby generating local mechanical stress on cells, and can further regulate cell behavior, resulting in cell interaction to promote osteogenesis, accelerate the repair process of bone defects under microgravity conditions, and achieve synergistic promotion of osteogenesis under microgravity conditions.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例中基于形状记忆材料的骨组织支架的制备方法流程图；FIG1 is a flow chart of a method for preparing a bone tissue scaffold based on a shape memory material according to an embodiment of the present invention;
图2为本发明实施例中骨缺损修复支架微创植入过程示意图；FIG2 is a schematic diagram of a minimally invasive implantation process of a bone defect repair scaffold according to an embodiment of the present invention;
图3为本发明实施例中三种骨组织支架的点云模型和三维视图；FIG3 is a point cloud model and a three-dimensional view of three bone tissue scaffolds according to an embodiment of the present invention;
图4为本发明实施例中不同孔隙率支架的压缩试验结果；FIG4 is a compression test result of scaffolds with different porosities according to an embodiment of the present invention;
图5为本发明实施例中支架在40℃、50℃、60℃温度下的压缩实验结果；FIG5 is a compression test result of the stent at 40° C., 50° C., and 60° C. in an embodiment of the present invention;
图6为本发明实施例中骨组织支架弹性模量随孔隙率和温度的预测结果；FIG6 is a prediction result of the elastic modulus of the bone tissue scaffold according to an embodiment of the present invention as a function of porosity and temperature;
图7为本发明实验例中为研究支架生物相容性和骨代谢能力进行的CCK-8试验的结果；FIG7 is the result of a CCK-8 test performed to study the biocompatibility and bone metabolism capacity of the scaffold in the experimental example of the present invention;
图8为本发明实验例的动物试验中术后取出股骨进行的影像学检查结果；FIG8 is the imaging examination result of the femur removed after surgery in the animal experiment of the present invention;
图9为本发明实验例中通过HE和MT染色评价支架植入后对股骨缺损的修复效果。FIG9 is an experimental example of the present invention, in which HE and MT staining are used to evaluate the repair effect of the stent on the femoral defect after implantation.
具体实施方式Detailed ways
组织工程技术的发展为受损组织的修复和重建提供了另一种选择。生物支架是组织工程研究的关键因素，可以作为模板指导组织再生。然而，即使有大量的缺陷填充物或临时支架作为临床产品使用，这些产品及其外科应用在技术、手术和制造和缩放等方面仍然存在不足。从结构角度来看，骨支架在修复骨缺损方面应该具备几个关键的能力或特性。首先，骨组织支架应具有一定的强度，以提供足够的支撑。其次，在微观层面上，骨组织支架应是具有一定连接通道的非均质多孔框架结构。这些通道有利于新骨组织的生长，是运输营养物质和代谢废物的关键，也是细胞分化信号的关键。第三，支架必须易于植入病变区域，例如通过微创植入。最后，支架应能很好地贴合骨缺损边界，促进成骨细胞的粘附和增殖。The development of tissue engineering technology has provided an alternative for the repair and reconstruction of damaged tissues. Biological scaffolds are a key factor in tissue engineering research and can serve as templates to guide tissue regeneration. However, even though there are a large number of defect fillers or temporary scaffolds used as clinical products, these products and their surgical applications still have deficiencies in terms of technology, surgery, manufacturing and scaling. From a structural perspective, bone scaffolds should have several key capabilities or characteristics in repairing bone defects. First, bone tissue scaffolds should have a certain strength to provide sufficient support. Second, at the microscopic level, bone tissue scaffolds should be heterogeneous porous framework structures with certain connecting channels. These channels are conducive to the growth of new bone tissue, are key to transporting nutrients and metabolic waste, and are key to cell differentiation signals. Third, the scaffold must be easy to implant into the lesion area, such as through minimally invasive implantation. Finally, the scaffold should be able to fit the bone defect boundary well and promote the adhesion and proliferation of osteoblasts.
目前，已有多种设计策略用于支架的制作。然而，这些支架仍然存在一些不可避免的问题，例如生物相容性差、制备效率低、力学性能差和孔隙率低等。许多支架采用水凝胶制备，但由于缺乏机械强度和固化时间不确定，通常导致支架与骨缺损的边缘接触不良。且一些用于制备水凝胶的单体或催化剂的细胞毒性也是不可忽视的。典型的原位水凝胶支架缺乏足够的孔隙度和相互连接的通道，这限制了细胞的迁移速度和水凝胶的生物降解。此外，对于生物活性陶瓷制备的支架，其脆性的力学性能和低孔隙连通性将极大地限制其应用。At present, there are many design strategies for the preparation of scaffolds. However, these scaffolds still have some inevitable problems, such as poor biocompatibility, low preparation efficiency, poor mechanical properties and low porosity. Many scaffolds are prepared using hydrogels, but due to the lack of mechanical strength and uncertain curing time, the scaffolds usually have poor contact with the edges of the bone defect. And the cytotoxicity of some monomers or catalysts used to prepare hydrogels cannot be ignored. Typical in situ hydrogel scaffolds lack sufficient porosity and interconnected channels, which limits the migration rate of cells and the biodegradation of hydrogels. In addition, for scaffolds prepared from bioactive ceramics, their brittle mechanical properties and low pore connectivity will greatly limit their applications.
临床上大多数骨支架由磷酸钙、聚酯或两者的组合组成。然而，很少有骨组织支架能同时满足上述几种特性。基于陶瓷或骨水泥的支架通常是脆弱的，这使得它们在植入过程中很容易破碎。以临床最常用的磷酸钙水泥支架为例，由于其刚性的力学特性，操作具有挑战性或效率低。多孔结构不容易被外科医生塑造或调整大小。最重要的是，该类型支架不能通过微创手术植入，仅适用于修复非承重或低承重部位的骨缺损。此外，如果支架在植入过程中掉落颗粒，颗粒会在髓腔之间漂移，导致异位骨形成，这大大增加了手术的难度和不确定性。同时，粒子的脱落在植入过程中似乎是不可避免的，因为支架必然会跟缺陷的边缘产生摩擦。Most bone scaffolds in clinical practice are composed of calcium phosphate, polyester, or a combination of the two. However, few bone tissue scaffolds can meet the above characteristics at the same time. Ceramic or bone cement-based scaffolds are usually fragile, which makes them easy to break during implantation. Taking the most commonly used calcium phosphate cement scaffold in clinical practice as an example, due to its rigid mechanical properties, the operation is challenging or inefficient. The porous structure is not easy to be shaped or resized by surgeons. Most importantly, this type of scaffold cannot be implanted through minimally invasive surgery and is only suitable for repairing bone defects in non-load-bearing or low-load-bearing areas. In addition, if the scaffold drops particles during implantation, the particles will drift between the medullary cavities, resulting in ectopic bone formation, which greatly increases the difficulty and uncertainty of the operation. At the same time, the shedding of particles seems to be inevitable during implantation because the scaffold will inevitably rub against the edge of the defect.
应力缺失会引发细胞骨架重塑、机械信号转导障碍及膜通透性减弱等一系列变化并最终抑制细胞分化、增殖和黏附，微重力环境影响细胞骨架的稳态，导致肌动蛋白网络重排，显著降低成骨细胞活性及成骨诱导因子合成，最终导致成骨缓慢。因此，微重力条件下会显著抑制力学-生物信号转换效率，最终抑制成骨。目前，尚缺乏微重力条件下补偿周期性应力的有效方案。Stress loss can trigger a series of changes such as cytoskeletal remodeling, mechanical signal transduction disorders, and reduced membrane permeability, and ultimately inhibit cell differentiation, proliferation, and adhesion. The microgravity environment affects the homeostasis of the cytoskeleton, leading to the rearrangement of the actin network, significantly reducing osteoblast activity and osteoinductive factor synthesis, and ultimately leading to slow osteogenesis. Therefore, microgravity conditions will significantly inhibit the efficiency of mechanical-biological signal conversion and ultimately inhibit osteogenesis. At present, there is still a lack of effective solutions to compensate for periodic stress under microgravity conditions.
本发明从几何学的角度对骨小梁的局部和全局形状进行描述，通过对骨组织内部多层级微结构的仿生设计，开发一种考虑微结构因素的4D打印仿生骨组织支架，改善支架的生物相容性，促进与周围骨组织的骨整合，对其进行力学性能分析并实现微创植入。The present invention describes the local and global shapes of trabeculae from a geometric perspective, and develops a 4D printed bionic bone tissue scaffold that takes microstructural factors into consideration through bionic design of the multi-level microstructure inside the bone tissue, thereby improving the biocompatibility of the scaffold, promoting bone integration with surrounding bone tissue, analyzing its mechanical properties, and achieving minimally invasive implantation.
为使本发明的上述目的、特征和优点能够更为明显易懂，下面结合附图对本发明的具体实施例做详细的说明。In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.
本发明实施例的一种基于形状记忆材料的骨组织支架的制备方法，包括以下步骤：A method for preparing a bone tissue scaffold based on a shape memory material according to an embodiment of the present invention comprises the following steps:
(1)以形状记忆聚合物、纳米颗粒、压电材料、Mxenes为原料制备形状记忆打印线材。(1) Shape memory printing filaments are prepared using shape memory polymers, nanoparticles, piezoelectric materials, and Mxenes as raw materials.
具体实施例中，将形状记忆聚合物、纳米颗粒、压电材料、Mxenes按照一定的配比溶解于溶剂中，混合均匀，待溶剂挥发得到固体原料，将固体原料切粒得到固体颗粒，利用挤出机将固体颗粒制备成打印线材。In a specific embodiment, shape memory polymer, nanoparticles, piezoelectric material, and Mxenes are dissolved in a solvent in a certain ratio and mixed evenly. The solvent is evaporated to obtain a solid raw material, the solid raw material is pelletized to obtain solid particles, and the solid particles are prepared into printing wires using an extruder.
其中，所述形状记忆聚合物为具有双向形状记忆行为的可降解热塑性形状记忆聚合物。示例性地，所述具有双向形状记忆行为的可降解热塑性形状记忆聚合物包括具有双向形状记忆效应的形状记忆聚氨酯、形状记忆聚乳酸、形状记忆聚己内酯、形状记忆壳聚糖的一种或多种以及它们的复合物和衍生物。Wherein, the shape memory polymer is a degradable thermoplastic shape memory polymer with bidirectional shape memory behavior. Exemplarily, the degradable thermoplastic shape memory polymer with bidirectional shape memory behavior includes one or more of shape memory polyurethane, shape memory polylactic acid, shape memory polycaprolactone, shape memory chitosan with bidirectional shape memory effect, and their composites and derivatives.
所述纳米颗粒包括Fe3O4纳米颗粒、纳米金颗粒、碳粉、炭黑、石墨烯、碳纳米管和镍粉中的一种或多种，掺杂的纳米颗粒的体积分数含量为1％-20％。通过调整纳米颗粒的体积分数，可以改善打印线材力学性能，得到具有响应功能的形状记忆打印线材。The nanoparticles include one or more ofFe3O4 nanoparticles,nanogold particles, carbon powder, carbon black, graphene, carbon nanotubes and nickel powder, and the volume fraction of the doped nanoparticles is 1%-20%. By adjusting the volume fraction of the nanoparticles, the mechanical properties of the printing wire can be improved to obtain a shape memory printing wire with a responsive function.
所述压电材料为钛酸钡、氧化锌纳米颗粒、铌酸钾钠和聚偏氟乙烯纳米材料中的一种或多种的混合物。所述压电材料的体积分数含量为1％-20％。The piezoelectric material is a mixture of one or more of barium titanate, zinc oxide nanoparticles, potassium sodium niobate and polyvinylidene fluoride nanomaterials. The volume fraction of the piezoelectric material is 1%-20%.
Mxenes是二维过渡金属碳化物和氮化物，其体积分数≤1％。Mxenes are two-dimensional transition metal carbides and nitrides with a volume fraction of ≤1%.
本实施例中，掺杂的纳米颗粒有助于通过远程驱动实现支架形变，通过环境刺激，可将形状记忆骨组织支架赋形为收缩状态，易于植入体内，且形状记忆骨组织支架由于形状记忆性能，能够在相应的刺激下回复其原状，并在骨缺损部位起到支撑作用。其中，外界环境为热、溶液、pH值、电、磁、电磁、光中的一种或多种因素。In this embodiment, the doped nanoparticles help to achieve the deformation of the scaffold through remote driving. Through environmental stimulation, the shape memory bone tissue scaffold can be shaped into a contracted state, which is easy to implant in the body, and the shape memory bone tissue scaffold can return to its original state under the corresponding stimulation due to its shape memory performance, and play a supporting role in the bone defect site. Among them, the external environment is one or more factors of heat, solution, pH value, electricity, magnetism, electromagnetism, and light.
内源性电场在胚胎发生、生理活动、伤口愈合、组织重塑等生物过程中起着指导作用，是维持细胞稳态所必需的。作为细胞外基质的生物物理线索，电场在促进在组织工程中具有重要的作用，与骨组织的生长、稳态、重塑和细胞代谢密切相关。本实施例的骨组织支架，在用于打印支架的线材中添加有压电材料和MXenes(二维过渡金属碳化物和氮化物)，使支架具有压电功能和摩擦电功能，在支架结构发生变形时产生微电流和电压，或者通过固(支架)液(体液)感应产生摩擦电，在支架结构和体液发生相对运动时产生微电流和电压。Endogenous electric fields play a guiding role in biological processes such as embryogenesis, physiological activities, wound healing, and tissue remodeling, and are necessary to maintain cell homeostasis. As a biophysical clue of the extracellular matrix, the electric field plays an important role in promoting tissue engineering, and is closely related to the growth, homeostasis, remodeling, and cell metabolism of bone tissue. The bone tissue scaffold of this embodiment has piezoelectric materials and MXenes (two-dimensional transition metal carbides and nitrides) added to the wire used to print the scaffold, so that the scaffold has piezoelectric and triboelectric functions, and generates microcurrent and voltage when the scaffold structure is deformed, or generates triboelectricity through solid (scaffold) liquid (body fluid) induction, and generates microcurrent and voltage when the scaffold structure and body fluid move relative to each other.
具体的，本实施例通过向形状记忆聚合物基体中添加Mxenes以增加支架的摩擦电性能。由于体液和添加了Mxenes的支架的得失电子能力不同，当支架和体液产生相对运动时，支架和体液之间会产生电势差，将驱动支架和体液中的电子在二者之间来回流动，从而产生电信号，以电诱导成骨细胞的分化和骨重塑，从而干预骨骼稳态，缓解骨质疏松症和骨质疏松症相关的骨折。Specifically, this embodiment adds Mxenes to the shape memory polymer matrix to increase the triboelectric properties of the stent. Since the body fluid and the stent with Mxenes added have different abilities to gain and lose electrons, when the stent and the body fluid move relative to each other, an electric potential difference will be generated between the stent and the body fluid, which will drive the electrons in the stent and the body fluid to flow back and forth between the two, thereby generating an electrical signal to electrically induce osteoblast differentiation and bone remodeling, thereby intervening in bone homeostasis and alleviating osteoporosis and osteoporosis-related fractures.
此外，在支架的制备过程中，通过在极化过程中使用高压电场来重新定向电畴，使整个支架保持净极化，导致极化的铁电体表现出压电活动。当支架受到可以改变支架极化水平的应力时，极化后的电偶极子极化方向改变，会导致支架的每个表面上出现电荷。同时，这些电荷会在电极表面产生相反的感应电荷。如果对支架施加压应力导致支架极化水平降低，则电极表面的自由电荷将在电负载上产生电流。相反，如果施加到支架上的拉伸应力导致支架极化水平升高并导致电流沿相反方向流动以平衡表面电荷。In addition, during the preparation of the scaffold, a high voltage electric field is used during the polarization process to redirect the electric domains so that the entire scaffold maintains a net polarization, causing the polarized ferroelectric to exhibit piezoelectric activity. When the scaffold is subjected to a stress that can change the polarization level of the scaffold, the polarization direction of the polarized electric dipole changes, resulting in charges appearing on each surface of the scaffold. At the same time, these charges will produce opposite induced charges on the electrode surface. If compressive stress is applied to the scaffold, resulting in a decrease in the polarization level of the scaffold, the free charges on the electrode surface will generate current on the electrical load. Conversely, if tensile stress is applied to the scaffold, the polarization level of the scaffold increases and causes current to flow in the opposite direction to balance the surface charge.
(2)根据骨组织材料的形态、结构，利用控制方程进行具有复杂分层空间建筑的骨组织支架的仿生设计，得到骨组织支架模型。(2) According to the morphology and structure of bone tissue materials, the control equation is used to carry out the bionic design of bone tissue scaffolds with complex layered spatial architecture to obtain a bone tissue scaffold model.
其中，仿生设计包括以下步骤：Among them, the bionic design includes the following steps:
利用计算机断层扫描获得骨小梁的模型，经过高斯滤波和阈值处理得到矿化骨相的三维二值图像；The trabecular bone model was obtained by computed tomography, and the three-dimensional binary image of the mineralized bone phase was obtained after Gaussian filtering and threshold processing;
随后利用移动立方体算法重构骨小梁表面的三角网格，并采用隐式曲面光顺对三角网格进行平滑处理；Then the triangular mesh of the trabecular surface was reconstructed using the marching cubes algorithm, and the triangular mesh was smoothed using implicit surface smoothing.
利用多尺度曲面拟合算法，在离散三角形网格的顶点估算出骨小梁表面的主曲率，将二阶多项式拟合到顶点周围的局部邻域进行曲率估计，并利用Minkowski函数来量化骨小梁的局部和全局几何特征；The principal curvature of the trabecular surface was estimated at the vertices of the discrete triangular mesh using a multiscale surface fitting algorithm, a second-order polynomial was fitted to the local neighborhood around the vertices for curvature estimation, and the Minkowski function was used to quantify the local and global geometric features of the trabecular bone.
结合极小曲面等几何建模方法，通过控制方程对支架进行仿生设计。Combined with geometric modeling methods such as minimal surfaces, the stent is biomimetically designed through control equations.
其中一些实施方式中，通过控制方程设计了可控孔隙率和孔隙形态的微结构单元。所述控制方程如公式(1-公式(3)所示。In some of the embodiments, a microstructure unit with controllable porosity and pore morphology is designed by a control equation. The control equation is shown in formula (1) to formula (3).
F1(x,y,z)＝α1 cos(x)+β1cos(y)+γ1 cos(z)+δ1 (1)。F1 (x, y, z) = α1 cos (x) + β1 cos (y) + γ1 cos (z) + δ1 (1).
F2(x,y,z)＝α2cos(x)sin(y)+β2cos(y)sin(z)+γ2cos(z)sin(x)+δ2 (2)。F2 (x, y, z) = α2 cos(x)sin(y) + β2 cos(y)sin(z) + γ2 cos(z)sin(x) + δ2 (2).
其中，Fi(x,y,z)表示第i个控制函数，i＝1,2,3，x、y、z分别表示笛卡尔坐标中的三个空间方向，αi表示第i个控制函数中的第一形状控制参数，βi表示第i个控制函数中的第二形状控制参数,γi表示第i个控制函数中的第三形状控制参数，λ表示第四形状控制参数，δi表示第i个控制函数中的控制实体比例的参数。Among them,Fi (x, y, z) represents the i-th control function, i = 1, 2, 3, x, y, z represent the three spatial directions in Cartesian coordinates respectively,αi represents the first shape control parameter in the i-th control function,βi represents the second shape control parameter in the i-th control function,γi represents the third shape control parameter in the i-th control function, λ represents the fourth shape control parameter, andδi represents the parameter of the control entity proportion in the i-th control function.
通过调整各参数的范围，可以得到不同构型和物理参数的骨组织支架。By adjusting the range of each parameter, bone tissue scaffolds with different configurations and physical parameters can be obtained.
本实施例中，骨组织支架的设计兼顾强度、孔隙率、孔径分布以及表面曲率等因素，并且能够针对不同的部位实现个性化定制及微创植入。通过对骨组织支架微结构的设计，从结构层面上增强了其生物活性和骨诱导性，能够促进其与周围骨组织的整合/结合以及成骨细胞的附着和分化，允许细胞迁移和组织浸润。In this embodiment, the design of the bone tissue scaffold takes into account factors such as strength, porosity, pore size distribution, and surface curvature, and can be customized and minimally invasively implanted for different parts. By designing the microstructure of the bone tissue scaffold, its biological activity and osteoinductivity are enhanced at the structural level, which can promote its integration/combination with surrounding bone tissue and the attachment and differentiation of osteoblasts, allowing cell migration and tissue infiltration.
(3)以所述形状记忆打印线材为原料，根据所述骨组织支架的模型，利用4D打印方法制备基于形状记忆的骨组织支架。(3) Using the shape memory printing wire as a raw material and according to the model of the bone tissue scaffold, a shape memory-based bone tissue scaffold is prepared by a 4D printing method.
其中一些实施方式中，所述4D打印技术为熔融沉积式打印、油墨直写打印或数字光处理打印等中的一种或多种。利用4D打印技术制备而成的形状记忆骨组织支架，具有分级多孔结构，且通过仿生设计而成的结构具有生物活性，能够促进与周围骨组织的整合以及成骨细胞的附着和分化。In some embodiments, the 4D printing technology is one or more of fused deposition printing, ink direct writing printing or digital light processing printing, etc. The shape memory bone tissue scaffold prepared by 4D printing technology has a hierarchical porous structure, and the structure designed by biomimetic design has biological activity, which can promote integration with surrounding bone tissue and the attachment and differentiation of osteoblasts.
其中一些实施方式中，采用形状记忆聚合物材料通过4D打印技术获得支架后，通过微创手术植入到骨缺损部位。植入过程示意如图XX所示，针对骨组织缺损(Bone TissueDefect)，首先利用CT扫描确定缺陷(Determination of defect by CT scan)，然后进行仿生设计(Bionics design)，4D打印(4Dprinting)支架，确定支架的临时形状(Temporaryshape)，将支架植入到缺陷中(Implanted into defect)，将支架恢复到磁性刺激下的原始形状(Recover original shape stimulated by magnetic)。In some embodiments, a shape memory polymer material is used to obtain a scaffold through 4D printing technology, and then the scaffold is implanted into the bone defect through minimally invasive surgery. The implantation process is shown in Figure XX. For bone tissue defects (Bone TissueDefect), the defect is first determined by CT scanning (Determination of defect by CT scan), and then bionic design (Bionics design), 4D printing (4Dprinting) the scaffold, the temporary shape (Temporaryshape) of the scaffold is determined, the scaffold is implanted into the defect (Implanted into defect), and the scaffold is restored to its original shape (Recover original shape stimulated by magnetic) under magnetic stimulation.
根据外界环境的改变，形状记忆聚合物基于玻璃态与橡胶态之间转变的形状记忆效应，使形状记忆骨组织支架进行形状的改变，并使其表现出自适应性，实现在远程非接触驱动条件下展开并与不规则缺陷完美匹配。如若形状记忆骨组织支架在植入体内之后由于操作问题造成支架与骨缺损界面匹配不理想，也可通过一定的环境刺激使其进行自我调整。此外，在具体应用过程中，通常在确定了骨缺损位置后，将骨组织支架植入相应位置，本实施例的骨组织支架也可在不确定骨缺损形状的状态下植入，利用其形状记忆效应，在玻璃化转变温度以上该材料处于较软的状态，在该状态下骨组织支架可自行填充不规则骨缺损，并与邻近组织完美的贴合。According to the changes in the external environment, the shape memory polymer changes the shape of the shape memory bone tissue scaffold based on the shape memory effect of the transition between the glass state and the rubber state, and makes it adaptive, so that it can be deployed under remote non-contact driving conditions and perfectly match the irregular defects. If the shape memory bone tissue scaffold does not match the interface between the scaffold and the bone defect well due to operational problems after being implanted in the body, it can also be self-adjusted through certain environmental stimuli. In addition, in the specific application process, the bone tissue scaffold is usually implanted in the corresponding position after the location of the bone defect is determined. The bone tissue scaffold of this embodiment can also be implanted in a state where the shape of the bone defect is uncertain. By utilizing its shape memory effect, the material is in a softer state above the glass transition temperature. In this state, the bone tissue scaffold can fill the irregular bone defect by itself and fit perfectly with the adjacent tissue.
本实施例制备的形状记忆骨组织支架，利用其形状记忆性能，可通过微创手术将体积压缩到极限的骨组织支架植入到体内，并在其达到骨缺损部位时，施加合适的刺激使其恢复到初始形状并适应不规则的缺陷，与相邻骨组织形成紧密接触以实现骨整合。另由于形状记忆骨组织支架具有适当的转变温度，在体温下具有足够的机械强度，因此还表现出优良的支撑性能。此外，利用形状记忆骨组织支架的双向形状记忆效应，可以在一定的刺激下进行循环往复的运动，该性能能够在微重力条件下通过往复形变为骨结构及细胞提供周期性轴向应力，成骨分化，同时促进血管生成，协同促进成骨。The shape memory bone tissue scaffold prepared in this embodiment can utilize its shape memory properties to implant the bone tissue scaffold compressed to the limit into the body through minimally invasive surgery, and when it reaches the bone defect site, apply appropriate stimulation to restore it to its original shape and adapt to the irregular defect, forming close contact with the adjacent bone tissue to achieve bone integration. In addition, since the shape memory bone tissue scaffold has an appropriate transition temperature and has sufficient mechanical strength at body temperature, it also exhibits excellent supporting performance. In addition, utilizing the bidirectional shape memory effect of the shape memory bone tissue scaffold, cyclic reciprocating motion can be performed under certain stimulation. This performance can provide periodic axial stress to bone structures and cells through reciprocating deformation under microgravity conditions, osteogenic differentiation, and promote angiogenesis at the same time, synergistically promoting osteogenesis.
其中一些实施方式中，在步骤(3)之前，还包括：对骨组织支架模型不同温度下的弹性模量进行预测，并进一步根据预测结果调整所述骨组织支架模型。在步骤(2)的仿生设计阶段，获得了不同孔隙率的骨组织支架，本步骤中，通过建立微分方法预测不同温度下支架的弹性模量，并根据预测结果调整模型设计阶段例如控制方程的各个参数，以调整支架的构型及物理参数等。In some embodiments, before step (3), the method further includes: predicting the elastic modulus of the bone tissue scaffold model at different temperatures, and further adjusting the bone tissue scaffold model according to the prediction results. In the bionic design stage of step (2), bone tissue scaffolds with different porosities are obtained. In this step, the elastic modulus of the scaffold at different temperatures is predicted by establishing a differential method, and the various parameters of the model design stage, such as the control equation, are adjusted according to the prediction results to adjust the configuration and physical parameters of the scaffold.
预测模型的建立过程如下：The process of establishing the prediction model is as follows:
SMP-PLA(形状记忆生物降解塑料聚乳酸)/Fe3O4的力学参数在玻璃化转变过程中发生了显著变化。当材料处于玻璃态时，链段被冻结，只有相对较小的运动单元能够运动。在橡胶态下，链段被激活，特别是在玻璃化转变温度附近，模量发生显著变化。通常用Williams-Landel-Ferry(WLF)方程来描述弹性模量与温度的关系。链段的运动能力通过松弛时间的长短来体现，即链段的运动能力越弱，其被拉伸的难度越大，相应的模量越大。因此，松弛模量随时间和温度的变化规律由WLF方程描述:The mechanical parameters of SMP-PLA (shape memory biodegradable plastic polylactic acid)/Fe3 O4 change significantly during the glass transition process. When the material is in the glassy state, the chain segments are frozen and only relatively small moving units can move. In the rubbery state, the chain segments are activated, especially near the glass transition temperature, and the modulus changes significantly. The Williams-Landel-Ferry (WLF) equation is usually used to describe the relationship between the elastic modulus and temperature. The mobility of the chain segment is reflected by the length of the relaxation time, that is, the weaker the mobility of the chain segment, the more difficult it is to be stretched, and the corresponding modulus is larger. Therefore, the change of the relaxation modulus with time and temperature is described by the WLF equation:
其中，Em为SMP-PLA/Fe3O4在温度“T”下的弹性模量，E0为SMP-PLA/Fe3O4在参考温度Tr下的弹性模量。C1和C2为WLF方程的拟合参数。Wherein,Em is the elastic modulus of SMP-PLA/Fe3 O4 at temperature “T”,E0 is the elastic modulus of SMP-PLA/Fe3 O4 at reference temperatureTr . C1 and C2 are the fitting parameters of the WLF equation.
对于多孔支架，孔隙率f是具有较高实际意义的参数之一。其中，支架的孔隙率为孔隙体积V与支架整体体积V0的百分比。For porous scaffolds, the porosity f is one of the parameters with high practical significance. The porosity of the scaffold is the percentage of the pore volume V to the overall volumeV0 of the scaffold.
由于孔隙分布较为均匀，支架等效于泡沫塑料，有效力学性能采用差分方案确定。根据微分格式，支架的体模量和剪切模量可表示为:Since the pore distribution is relatively uniform, the scaffold is equivalent to foam plastic, and the effective mechanical properties are determined by differential scheme. According to the differential format, the bulk modulus and shear modulus of the scaffold can be expressed as:
dμ*/df＝-[1/(1-f)]·μ*·[15·(1-ν*)/(7-5·ν*)] (6)dμ* /df=-[1/(1-f)]·μ* ·[15·(1-ν* )/(7-5·ν* )] (6)
dK*/df＝-[1/(1-f)]·K*·[3K*/4μ*+1] (7)dK* /df=-[1/(1-f)]·K* ·[3K* /4μ* +1] (7)
其中，μ*为支架的有效剪切模量，ν*为有效泊松比，K*是有效体积模量，f为孔隙率。假设支架结构是各向同性的，各参数的关系为：Among them, μ* is the effective shear modulus of the scaffold, ν* is the effective Poisson's ratio, K* is the effective bulk modulus, and f is the porosity. Assuming that the scaffold structure is isotropic, the relationship between the parameters is:
ν*＝(3K*-2μ*)/(6K*+2μ*)；ν* =(3K* -2μ* )/(6K* +2μ* );
初始体积模量和剪切模量定义为Km和μm。利用初始条件f＝0,μ*＝μm和K*＝Km。μ*、K*和f可以进一步描述为：The initial bulk modulus and shear modulus are defined as Km and μm . Using the initial conditions f = 0, μ* = μm and K* = Km . μ* , K* and f can be further described as:
dμ*/df＝-5μ*(3K*+4μ*)/[(1-f)(9K*+8μ*)] (8)dμ* /df=-5μ* (3K* +4μ* )/[(1-f)(9K* +8μ* )] (8)
消去f和df项，方程可以进一步表示为：Eliminating the f and df terms, the equation can be further expressed as:
dK*/dμ＝(K*/μ*)(9K*+8μ*)/20μ* (9)dK* /dμ=(K* /μ* )(9K* +8μ* )/20μ* (9)
进一步，支架的杨氏模量可确定为:Further, the Young's modulus of the scaffold can be determined as:
E*＝9K*μ*/(3K*+μ*)＝36Kmμ*/[15Km+(4μm-3Km)(μ*/μm)0.6] (11)E* = 9K* μ* /(3K* +μ* ) = 36Kmμ*/ [15Km +(4μm -3Km )(μ* /μm )0.6 ] (11)
基体材料在整个结构中的比例被定义为则可得到微分方程的解:The proportion of matrix material in the entire structure is defined as Then we can get the solution of the differential equation:
根据反函数理论，体积模量和弹性模量可以唯一地表示为:According to the inverse function theory, the bulk modulus and elastic modulus can be uniquely expressed as:
为使支架与孔隙率的关系更清晰明了，弹性模量与孔隙率的近似微分方程可表示为:To make the relationship between the scaffold and porosity clearer, the approximate differential equation of elastic modulus and porosity can be expressed as:
令方程可进一步表示为：make The equation can be further expressed as:
通过积分，可进一步表示为:Through integration, it can be further expressed as:
孔隙度的连续可微函数G1(f)满足初始条件:The continuously differentiable function of porosity G1 (f) satisfies the initial condition:
F(0)＝0,F(1)＝+∞ (18)F(0)＝0,F(1)＝+∞ (18)
因此，G(f)可用多项式近似表示为：Therefore, G(f) can be approximated by a polynomial as:
G(f)＝A1f+A2f2+...Anfn (19)G(f)＝A1f +A2f2+ ...Anfn (19 )
误差可以通过残差项估计:The error can be estimated by the residual term:
假设n→∞，幂函数级数一致收敛于G(f)，并得到了弹性模量的理论解。将函数G(f)近似为有限级数，可得到支架弹性模量的指数预测公式。其中，双参数计算模型可表示为:Assuming n→∞, the power function series converges to G(f) uniformly, and the theoretical solution of the elastic modulus is obtained. Approximating the function G(f) as a finite series, the exponential prediction formula of the stent elastic modulus can be obtained. Among them, the dual-parameter calculation model can be expressed as:
根据三种控制方程(公式(1)-公式(3))通过4D打印技术分别获得三种结构的骨组织支架，通过实验和上述仿真获得支架的弹性模量随孔隙率的变化如图6所示。可以看出，孔隙率的大小对支架力学性能影响较大，相同孔隙率的支架力学性能之间无明显差异。According to the three control equations (Formula (1)-Formula (3)), three structures of bone tissue scaffolds were obtained by 4D printing technology. The elastic modulus of the scaffolds obtained by experiments and the above simulations varies with the porosity as shown in Figure 6. It can be seen that the porosity has a great influence on the mechanical properties of the scaffolds, and there is no obvious difference in the mechanical properties of scaffolds with the same porosity.
本实施例制备的基于形状记忆材料的骨组织支架，为多尺度/多层级的梯度功能结构，符合自然骨孔隙率和孔径由外到内呈梯度变化的规律，其孔隙率为10％-90％，其抗压强度为1MPa-50MPa。由于具有功能特性的梯度设计特点，因此可以通过不同部位使用不同玻璃化转变温度的材料，使得该结构在不同的时间选择不同的驱动方式驱动支架结构分级展开。The bone tissue scaffold based on shape memory material prepared in this embodiment is a multi-scale/multi-level gradient functional structure, which conforms to the law that the porosity and pore size of natural bones change gradually from the outside to the inside, and its porosity is 10%-90%, and its compressive strength is 1MPa-50MPa. Due to the gradient design characteristics with functional characteristics, materials with different glass transition temperatures can be used in different parts, so that the structure can select different driving modes at different times to drive the scaffold structure to unfold in stages.
综上，本实施例制备的骨组织支架由于采用具有双向形状记忆效应的形状记忆聚合物制备而成，可以在热、溶液、pH值、电、磁、电磁、光等中的一种或多种刺激方式下循环往复运动。例如，通过对骨组织支架施加交变磁场，利用其双向形状记忆效应可实现往复形变，可以在微重力条件下提供成骨所需的周期性应力补偿，进而对细胞产生局部机械应力，可进一步调节细胞行为，导致的细胞交互促进成骨，加速骨缺损在微重力条件下的修复过程。In summary, the bone tissue scaffold prepared in this embodiment is made of a shape memory polymer with a two-way shape memory effect, and can be cyclically reciprocated under one or more stimulation modes of heat, solution, pH value, electricity, magnetism, electromagnetism, light, etc. For example, by applying an alternating magnetic field to the bone tissue scaffold, reciprocating deformation can be achieved by utilizing its two-way shape memory effect, and the periodic stress compensation required for osteogenesis can be provided under microgravity conditions, thereby generating local mechanical stress on cells, which can further regulate cell behavior, resulting in cell interaction promoting osteogenesis and accelerating the repair process of bone defects under microgravity conditions.
本实施例制备了一种“自适应”功能的基于形状记忆聚合物的4D打印电活性多孔骨组织支架。形状记忆材料是一种具有主动变形能力的智能材料，它可以在热、电、磁或光的刺激下产生不同的形状，并恢复到原始形状。该形状记忆支架可以通过微创手术在高度压实的状态下植入目标位置，并在磁场等刺激下实现缺损填充。同时，该支架可以为骨缺损提供足够的支撑。此外，利用形状记忆聚合物的双向形状记忆行为，能够在一定的刺激条件下完成非接触式往复形变，具有形变可逆、频率可控、条件温和耐疲劳的特性，有助于在微重力条件下提供成骨所需的周期性应力。同时利用压电和摩擦电构筑体内的微电环境，促进骨细胞的生长和缺损的修复。本发明形状记忆骨组织支架具有巨大的临床转化潜力。This embodiment prepares a 4D printed electroactive porous bone tissue scaffold based on shape memory polymer with "adaptive" function. Shape memory material is an intelligent material with active deformation ability. It can produce different shapes under the stimulation of heat, electricity, magnetism or light, and restore to the original shape. The shape memory scaffold can be implanted in the target position in a highly compacted state through minimally invasive surgery, and the defect can be filled under the stimulation of magnetic field and the like. At the same time, the scaffold can provide sufficient support for the bone defect. In addition, by utilizing the bidirectional shape memory behavior of the shape memory polymer, non-contact reciprocating deformation can be completed under certain stimulation conditions, and it has the characteristics of reversible deformation, controllable frequency, mild conditions and fatigue resistance, which helps to provide the periodic stress required for osteogenesis under microgravity conditions. At the same time, piezoelectricity and triboelectricity are used to construct a microelectric environment in the body to promote the growth of bone cells and the repair of defects. The shape memory bone tissue scaffold of the present invention has great clinical transformation potential.
下面通过具体实施例对本发明进一步说明。The present invention is further described below by means of specific examples.
实施例1Example 1
将形状记忆聚合物、纳米颗粒、压电材料、Mxenes按照一定的配比溶解于溶剂中，混合均匀，待溶剂挥发得到固体原料，将固体原料切粒得到固体颗粒，利用挤出机将固体颗粒制备成打印线材。其中，形状记忆聚合物包括形状记忆聚氨酯、形状记忆聚乳酸、形状记忆聚己内酯、形状记忆壳聚糖及其复合物和衍生物中的一种或多种。纳米颗粒包括Fe3O4纳米颗粒、纳米金颗粒、碳粉、炭黑、石墨烯、碳纳米管和镍粉中的一种或多种。压电材料为钛酸钡、氧化锌纳米颗粒、铌酸钾钠和聚偏氟乙烯纳米材料中的一种或多种的混合物。纳米颗粒的体积分数含量为1％-20％。压电材料的体积分数含量为1％-20％。Mxenes的其体积分数≤1％。Shape memory polymer, nanoparticles, piezoelectric material, and Mxenes are dissolved in a solvent according to a certain ratio, mixed evenly, and solid raw materials are obtained after the solvent evaporates. The solid raw materials are pelletized to obtain solid particles, and the solid particles are prepared into printing wires by an extruder. Among them, the shape memory polymer includes one or more of shape memory polyurethane, shape memory polylactic acid, shape memory polycaprolactone, shape memory chitosan, and their complexes and derivatives. Nanoparticles include one or more of Fe3 O4 nanoparticles, nanogold particles, carbon powder, carbon black, graphene, carbon nanotubes, and nickel powder. The piezoelectric material is a mixture of one or more of barium titanate, zinc oxide nanoparticles, potassium sodium niobate, and polyvinylidene fluoride nanomaterials. The volume fraction of nanoparticles is 1%-20%. The volume fraction of piezoelectric materials is 1%-20%. The volume fraction of Mxenes is ≤1%.
根据骨组织材料的形态、结构，利用控制方程(公式(1))进行骨组织支架的仿生设计，得到骨组织支架的模型；According to the morphology and structure of the bone tissue material, the control equation (Formula (1)) is used to perform the bionic design of the bone tissue scaffold and obtain the model of the bone tissue scaffold;
以所述形状记忆打印线材为原料，根据所述骨组织支架的模型，利用4D打印方法制备直径为8mm的基于形状记忆的骨组织支架，记为支架S-I。Using the shape memory printing wire as raw material and according to the model of the bone tissue scaffold, a shape memory-based bone tissue scaffold with a diameter of 8 mm was prepared by a 4D printing method, which was recorded as scaffold S-I.
实施例2Example 2
本实施例与实施例1的区别在于，采用控制方程(公式(2))进行仿生设计，最终获得的支架记为支架S-II。The difference between this embodiment and embodiment 1 is that the control equation (formula (2)) is used for bionic design, and the bracket finally obtained is recorded as bracket S-II.
实施例3Example 3
本实施例与实施例1的区别在于，采用控制方程(公式(3))进行仿生设计，最终获得的支架记为支架S-III。The difference between this embodiment and embodiment 1 is that the control equation (formula (3)) is used for bionic design, and the bracket finally obtained is recorded as bracket S-III.
实施例1-3制备的支架的点云模型由Python获取，如图3A所示。通过开发的脚本程序将模型导入Rhino软件，赋予其厚度，设计的孔隙率为60％的三种支架的三维视图如图3B所示。The point cloud models of the scaffolds prepared in Examples 1-3 were obtained by Python, as shown in Figure 3A. The models were imported into Rhino software through the developed script program, and given thickness. The three-dimensional views of the three scaffolds with a designed porosity of 60% are shown in Figure 3B.
分别制备具有不同孔隙率的支架，如制备孔隙率为50％、60％、70％的支架S-I，制备孔隙率为50％、60％、70％的支架S-II，制备孔隙率为50％、60％、70％的支架S-III。不同孔隙率支架的压缩试验结果如图4所示。当孔隙率为70％、60％和50％时，支架在37℃下的弹性模量和抗压强度分别约为150MPa和14MPa,260MPa和20MPa,350MPa和25MPa，如图4所示。需要说明的是，由于支架的力学性能跟结构关系不大，主要是受孔隙率的影响，因此三种支架具有类似的力学性能，这里主要说明的是不同孔隙率下支架的力学性能，因此上文中孔隙率为70％、60％和50％时的支架弹性模量和抗压强度可以是三个支架中的任一个，也因此用“约”来描述不同孔隙率下支架的弹性模量和抗压强度。Scaffolds with different porosities were prepared, such as scaffold S-I with porosities of 50%, 60%, and 70%, scaffold S-II with porosities of 50%, 60%, and 70%, and scaffold S-III with porosities of 50%, 60%, and 70%. The compression test results of scaffolds with different porosities are shown in Figure 4. When the porosity is 70%, 60%, and 50%, the elastic modulus and compressive strength of the scaffold at 37°C are approximately 150MPa and 14MPa, 260MPa and 20MPa, and 350MPa and 25MPa, respectively, as shown in Figure 4. It should be noted that since the mechanical properties of the scaffold have little to do with the structure and are mainly affected by the porosity, the three scaffolds have similar mechanical properties. What is mainly described here is the mechanical properties of the scaffolds at different porosities. Therefore, the elastic modulus and compressive strength of the scaffolds at porosities of 70%, 60% and 50% in the above text can be any of the three scaffolds, and therefore "approximately" is used to describe the elastic modulus and compressive strength of the scaffolds at different porosities.
对于孔隙率为60％的支架进行了不同温度下的力学性能测试。不同温度下支架的应力-应变曲线如图5所示。其中，A图代表支架S-I在不同温度下的压缩性能，B图代表支架S-II在不同温度下的压缩性能，C图代表支架S-III在不同温度下的压缩性能。可以看出，不管是哪个支架，支架结构在40℃时具有较高的强度，而在50℃和60℃时，屈服强度急剧下降。因此，当温度较低时，支架的刚度和强度较高，可以为骨缺损提供一定的支撑。当温度较高时，支架是柔软的，这大大方便了成型过程，并将支架压缩到最小规模。支架的可变刚度和形状记忆特性使得微创手术得以成功实施。Mechanical properties tests were carried out at different temperatures for the scaffold with a porosity of 60%. The stress-strain curves of the scaffolds at different temperatures are shown in Figure 5. Among them, Figure A represents the compression performance of the scaffold S-I at different temperatures, Figure B represents the compression performance of the scaffold S-II at different temperatures, and Figure C represents the compression performance of the scaffold S-III at different temperatures. It can be seen that no matter which scaffold, the scaffold structure has higher strength at 40°C, while the yield strength drops sharply at 50°C and 60°C. Therefore, when the temperature is low, the stiffness and strength of the scaffold are high, which can provide certain support for the bone defect. When the temperature is high, the scaffold is soft, which greatly facilitates the molding process and compresses the scaffold to the smallest scale. The variable stiffness and shape memory properties of the scaffold enable minimally invasive surgery to be successfully implemented.
实验例1Experimental Example 1
通过细胞实验对孔隙率为60％的支架进行体内外成骨实验。具体实验方案如下：Through cell experiments, in vitro and in vivo osteogenesis experiments were conducted on scaffolds with a porosity of 60%. The specific experimental plan is as follows:
为了研究支架对细胞活性的促进作用，本实施例进行活死细胞染色和CCK-8试验。结果如图7所示，其中，A图为CCK-8测定共培养4h、8h、12h后成骨细胞的短期生物相容性；B图为CCK-8测定共培养1d、3d后成骨细胞的OD值，7d后共培养，考察长期的生物相容性；C图为成骨效果评价，D图为培养10d后研究成骨分化相关基因的表达:RUNx2、OCN和ALP。结果显示，支架S-I和支架S-III在12h时附着的细胞数量较4小时时明显增加(图7中A图)。此外，与仅培养1天的细胞相比，支架S-I、S-II和S-III上的细胞数量在第3天和第7天显著增加(图7中B图)。可见，支架对细胞增殖具有良好的诱导作用。In order to study the promotion of scaffold to cell activity, the present embodiment carries out live and dead cell staining and CCK-8 test.As shown in Figure 7, A figure is CCK-8 to measure the short-term biocompatibility of osteoblasts after co-cultivation 4h, 8h, 12h; B figure is CCK-8 to measure the OD value of osteoblasts after co-cultivation 1d, 3d, co-cultivation after 7d, and investigates long-term biocompatibility; C figure is osteogenesis effect evaluation, and D figure is to study the expression of osteogenic differentiation-related genes after culturing 10d: RUNx2, OCN and ALP.As a result, the number of cells attached to scaffold S-I and scaffold S-III at 12h is significantly increased compared with 4 hours (Fig. A in Fig. 7).In addition, compared with cells cultured only for 1 day, the number of cells on scaffolds S-I, S-II and S-III significantly increased on the 3rd and 7th days (Fig. B in Fig. 7).It can be seen that scaffold has a good induction effect on cell proliferation.
与支架共培养2d后，用Calcein-AM和碘化丙啶对的细胞质进行染色。结果显示，S-I、S-II和S-III组细胞除对照组(Control)中少数细胞外，均未被染红。由此可见，成骨细胞在三种支架上存活率较高，且与支架表面粘附良好，说明支架具有良好的生物相容性。After 2 days of co-culture with the scaffold, the cytoplasm was stained with Calcein-AM and propidium iodide. The results showed that the cells in the S-I, S-II and S-III groups were not stained red except for a few cells in the control group (Control). It can be seen that the osteoblasts have a high survival rate on the three scaffolds and adhere well to the scaffold surface, indicating that the scaffold has good biocompatibility.
通过分析接种在支架上的成骨细胞的活性和形态，进一步评价其生物相容性。碱性磷酸酶(ALP)可作为成骨细胞分化和矿化的早期指标，通过ALP活性实验研究支架的骨代谢潜能。实验分为4组，分别为单独在培养基中培养的对照组和与细胞共培养的3个实验组。培养7d后，四组ALP活性检测结果均无统计学意义。但在第14天，S-I、S-II和S-III组ALP活性显著升高，如图7中C图所示。The biocompatibility was further evaluated by analyzing the activity and morphology of osteoblasts seeded on the scaffold. Alkaline phosphatase (ALP) can be used as an early indicator of osteoblast differentiation and mineralization. The bone metabolism potential of the scaffold was studied by ALP activity experiment. The experiment was divided into 4 groups, namely a control group cultured alone in culture medium and 3 experimental groups co-cultured with cells. After 7 days of culture, the ALP activity test results of the four groups were not statistically significant. However, on the 14th day, the ALP activity of the S-I, S-II and S-III groups increased significantly, as shown in Figure 7C.
通过Rt-PCR分析不同骨形成基因的相对表达量，评价支架的成骨能力。在成骨细胞分化的早期，ALP的表达水平会升高，而runt相关转录因子2(RUNx2)是通过与骨细胞结合而触发ALP表达的最重要的转录因子。培养14d后，3种支架对成骨代谢产物的表达均有不同程度的促进作用，但均优于对照组。S-I、S-II、S-III组RUNx2、OCN、ALP相对表达量与对照组比较，差异均有统计学意义(图7中D图)。与对照组相比，S-II组4个基因的相对表达量分别为2.8、2.1、0.29、2.324。由此可见，三种支架的孔隙率和最小微表面的分布均具有良好的促成骨作用。The relative expression of different bone formation genes was analyzed by RT-PCR to evaluate the osteogenic ability of the scaffold. In the early stage of osteoblast differentiation, the expression level of ALP increases, and runt-related transcription factor 2 (RUNx2) is the most important transcription factor that triggers ALP expression by binding to osteocytes. After 14 days of culture, the three scaffolds promoted the expression of osteogenic metabolites to varying degrees, but all were better than the control group. The relative expression of RUNx2, OCN, and ALP in the S-I, S-II, and S-III groups was significantly different from that in the control group (Figure 7, Figure D). Compared with the control group, the relative expression of the four genes in the S-II group was 2.8, 2.1, 0.29, and 2.324, respectively. It can be seen that the porosity and the distribution of the minimum microsurface of the three scaffolds have a good osteogenic effect.
实验例2Experimental Example 2
通过动物实验对孔隙率为60％的支架进行了动物实验。本实验选用雄性大白兔，由哈尔滨医科大学动物实验中心提供，所有动物实验操作均遵守哈尔滨医科大学动物实验伦理规定。动物实验的具体操作步骤如下：Animal experiments were conducted on scaffolds with a porosity of 60%. Male white rabbits were used in this experiment, which were provided by the Animal Experiment Center of Harbin Medical University. All animal experimental operations complied with the animal experiment ethics regulations of Harbin Medical University. The specific operating steps of the animal experiment are as follows:
实验分为一个对照组和四个实验组，对照组(Control)：人工骨；S-I组:压实支架S-I植入并在磁场中展开；S-II组:压实支架S-II植入并展开于磁场中；S-III组：植入压实支架S-III并在磁场中展开；S-IV组：将原直径8mm的支架S-II压缩至6.5mm，直接植入6.5mm缺损处，无需展开手术。The experiment was divided into a control group and four experimental groups: Control group (Control): artificial bone; S-I group: compacted stent S-I was implanted and unfolded in a magnetic field; S-II group: compacted stent S-II was implanted and unfolded in a magnetic field; S-III group: compacted stent S-III was implanted and unfolded in a magnetic field; S-IV group: the original 8mm diameter stent S-II was compressed to 6.5mm and directly implanted into the 6.5mm defect without the need for surgery.
在兔股骨远端引入直径为6.5mm的骨缺损，在骨缺损模型中植入压缩直径为3mm的支架S-I、S-II和S-III。对于S-IV组，其是将原直径8mm的支架S-II压缩至6.5mm，并直接植入6.5mm缺损处，无需展开手术。在磁场刺激下，支架S-I、S-II和S-III逐渐恢复到初始形状，并很好地填充了骨缺损。由于支架直径比骨缺损直径大2mm，支架充分展开后，支架表面与骨缺损之间存在一定的预紧力，进一步增强了固定效果。此外，支架的直径比骨缺损的直径大，更有利于填充不规则的骨缺损。术后取出股骨进行影像学检查，如图8所示，其中A图为12周时兔股骨缺损切片的Micro-CT图像，B图为Tb/Sp、BV和BV/TV定量分析，数值以均值±SD表示，**P<0.01，***P<0.001，****P<0.0001。此外，还进行苏木素-伊红(HE)染色和马松三色(MT)染色，HE和MT染色评价支架植入后对股骨缺损的修复效果如图9所示。A bone defect with a diameter of 6.5 mm was introduced into the distal end of the rabbit femur, and the scaffolds S-I, S-II and S-III with a compressed diameter of 3 mm were implanted in the bone defect model. For the S-IV group, the scaffold S-II with an original diameter of 8 mm was compressed to 6.5 mm and directly implanted into the 6.5 mm defect without the need for surgery. Under magnetic field stimulation, the scaffolds S-I, S-II and S-III gradually returned to their initial shape and filled the bone defect well. Since the scaffold diameter is 2 mm larger than the bone defect diameter, there is a certain pre-tightening force between the scaffold surface and the bone defect after the scaffold is fully deployed, which further enhances the fixation effect. In addition, the diameter of the scaffold is larger than the diameter of the bone defect, which is more conducive to filling irregular bone defects. The femur was removed after surgery for imaging examination, as shown in Figure 8, where Figure A is a Micro-CT image of the rabbit femoral defect section at 12 weeks, and Figure B is a quantitative analysis of Tb/Sp, BV, and BV/TV, with values expressed as mean ± SD, **P < 0.01, ***P < 0.001, ****P < 0.0001. In addition, hematoxylin-eosin (HE) staining and Masson's trichrome (MT) staining were performed, and the HE and MT staining were used to evaluate the repair effect of the femoral defect after stent implantation, as shown in Figure 9.
第4周时，无论HE染色还是MT染色结果，实验组的修复效果均优于对照组。8周时，Micro-CT分析显示支架S-II表现出持续的成骨作用，骨缺损得到修复。试验组的BV和BV/TV均高于对照组。HE和MT染色显示，实验组骨组织成熟度明显提高，胶原纤维增多。此外，在支架S-I、S-II、S-III、S-IV中观察到组织修复。结果进一步表明，该支架具有较高的骨诱导性，并能促进组织生长。植入后12周，Micro-CT定量分析显示，对照组与实验组骨体积差异无统计学意义。但试验组BV、BV/TV均显著高于对照组。与对照组相比，本发明实施例制备的三种支架均表现出良好的修复效果，且髓腔内未出现矿化骨。组织学验证进一步证实了该结构的修复效果，如图9所示。At the 4th week, the repair effect of the experimental group was better than that of the control group, regardless of the results of HE staining or MT staining. At 8 weeks, Micro-CT analysis showed that the scaffold S-II showed a sustained osteogenesis and the bone defect was repaired. The BV and BV/TV of the experimental group were higher than those of the control group. HE and MT staining showed that the maturity of the bone tissue in the experimental group was significantly improved, and the number of collagen fibers increased. In addition, tissue repair was observed in scaffolds S-I, S-II, S-III, and S-IV. The results further showed that the scaffold had high osteoinductivity and could promote tissue growth. 12 weeks after implantation, Micro-CT quantitative analysis showed that there was no statistically significant difference in bone volume between the control group and the experimental group. However, the BV and BV/TV of the experimental group were significantly higher than those of the control group. Compared with the control group, the three scaffolds prepared in the embodiment of the present invention all showed good repair effects, and no mineralized bone appeared in the medullary cavity. Histological verification further confirmed the repair effect of the structure, as shown in Figure 9.
虽然本发明披露如上，但本发明的保护范围并非仅限于此。本领域技术人员在不脱离本发明的精神和范围的前提下，可进行各种变更与修改，这些变更与修改均将落入本发明的保护范围。Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.
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