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4D-printed dual-responsive bioscaffolds for treating critical-sized irregular bone defects
用于治疗临界尺寸不规则骨缺损的 4D 打印双响应生物支架

Yangyang , Jiaqian You , Huixin , Chong Wang , Shaobo Zhai , Sicong Ren ,
Yangyang , Jiaqian You , Huixin , Chong Wang , Shaobo Zhai , Sicong Ren
Xiuyu Liu , Yidi Zhang , Yanmin Zhou ,
Xiuyu Liu , Yidi Zhang , Yanmin Zhou
Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling, Hospital of Stomatology, Jilin University, Changchun 130021, Jilin, China
吉林大学口腔医院牙齿发育与骨重塑吉林省重点实验室,吉林长春 130021
School of Stomatology, Jilin University, Changchun 130021, Jilin, China
吉林大学口腔医学院,吉林长春 130021
c School of Mechanical Engineering, Dongguan University of Technology, Dongguan 523808, Guangdong, China
c 东莞理工大学机械工程学院,中国广东东莞 523808

A R T I C L E I N F O

Keywords: 关键词:

print  打印
Bone regeneration 骨再生
Magnetic stimulation 磁刺激
Thermal stimulation 热刺激

Abstract 摘要

A B S T R A C T Currently, treating irregular bone defects with a critical size is still challenging as material implantation in the complex defects with irregular shapes, effective osteogenesis and sufficient vascularization cannot be easily achieved simultaneously via a simple strategy. Herein, a dual-responsive bone tissue engineering scaffold is developed using a 4D printing strategy by intergrating a single type of multifunctional magnetic nanoparticles, , with printing inks made of bioceramics and biopolymers. Minimally invasive implantation, fluent navigation of scaffolds as well as the precise boundary matching between scaffolds and the irregular defects are achieved through near-infrared (NIR) irradiation-based temperature responsive shape recovery and static magnetic field (SMF) stimulation. Moreover, improved bone regeneration in critical-sized bone defects is successfully achieved through the activation of PI3K/AKT pathway which significantly promotes osteogenic differentiation and vascularization. Besides, NIR-based photothermal stimulation upregulates the expression of heat shock protein (HSP90) and further promotes the osteogenesis and vascularization. The in vivo study also confirms that our scaffold can precisely fit the bone defect, and induce satisfactory osteogenesis and angiogenesis. This work provides a facile strategy to simultaneously-realize easy scaffold implantation in irregular bone defects and improves osteogenesis and angiogenesis by integrating a single type of multifunctional nanoparticles with scaffold matrices.
A B S T R A C T 目前,治疗具有临界尺寸的不规则骨缺损仍具有挑战性,因为通过简单的策略难以同时实现在形状不规则的复杂缺损中植入材料、有效成骨和充分血管化。本文采用 4D 打印策略,将单一类型的多功能磁性纳米粒子 与生物陶瓷和生物聚合物制成的打印墨水相融合,开发出了一种双重响应骨组织工程支架。通过基于近红外(NIR)辐照的温度响应形状恢复和静态磁场(SMF)刺激,实现了微创植入、支架的流畅导航以及支架与不规则缺陷之间的精确边界匹配。此外,通过激活 PI3K/AKT 通路,显著促进成骨分化和血管化,成功改善了临界大小骨缺损的骨再生。此外,基于近红外的光热刺激可上调热休克蛋白(HSP90)的表达,进一步促进骨生成和血管化。体内研究也证实了我们的支架能精确地贴合骨缺损,并诱导令人满意的成骨和血管生成。这项工作提供了一种简便的策略,通过将单一类型的多功能纳米颗粒与支架基质相结合,同时实现支架在不规则骨缺损中的简便植入,并改善成骨和血管生成。

1. Introduction 1.导言

The increasing number of diseases such as trauma, tumors and infections, which are often treated with irregular bone defects, has led to a surge in the demand for tissue-engineered bone [1]. Although autologous bone grafting is the gold standard for clinical treatment of bone defects, it suffers from donor shortage, secondary trauma at the donor site and difficulty in adapting to the defect site [2]. Bone tissue engineering scaffolds with customized shape and architecture can be produced by 3D printers to treat critical-sized bone defect when a specially designed digital STL file was already employed [3]. However, traumatic and bone disease related bone defects often provide surgeons with a complex implantation environment. For instance, surgical space in maxillary sinus floor elevation is normally very narrow, while the bone defect has an irregular shape. In such a case, conventional 3D printed scaffolds are difficult to maintain its initial shape during the implantation process, and the forcible implantation may either damage the scaffold or cause a secondary trauma [4]. Hence, there is an urgent demand to develop a shape-adaptive scaffold which is capable of minimally invasive implantation and stimulate both bone tissue and blood vessel regeneration, effectively.
外伤、肿瘤和感染等疾病越来越多,而这些疾病的治疗往往需要不规则的骨缺损,因此对组织工程骨的需求激增[1]。虽然自体骨移植是临床治疗骨缺损的金标准,但它存在供体短缺、供体部位二次创伤以及难以适应缺损部位等问题[2]。如果已经采用了专门设计的数字 STL 文件,则可通过三维打印机制造出具有定制形状和结构的骨组织工程支架,用于治疗临界尺寸的骨缺损[3]。然而,与创伤和骨病相关的骨缺损往往会给外科医生带来复杂的植入环境。例如,上颌窦底隆起的手术空间通常非常狭窄,而骨缺损的形状不规则。在这种情况下,传统的 3D 打印支架很难在植入过程中保持其初始形状,强行植入可能会损坏支架或造成二次创伤[4]。因此,人们迫切需要开发一种形状自适应支架,既能进行微创植入,又能有效刺激骨组织和血管再生。
The emergence of 4D printing offers a potent solution to make such scaffolds for treating irregular bone defect [5]. The concept of 4D printing is to add a temporal dimension to printing so that the shape and morphology of the printed scaffold changes in response to specific stimuli [6]. 4D printing is originally derived from 3D printing of shape memory materials (SMMs). Poly lactic-co-glycolic acid (PLGA) is a biodegradable SMM which is widely employed in the biomedical field and was approved by FDA [7]. PLGA not only possesses excellent biocompatibility, but also can regulate the glass transition temperature (Tg) of the polymer to be close to the acceptable temperature for the human body through a change in the ratio of lactic acid and glycolic acid [8]. In recent years, temperature-responsive SMMs integrated with photothermal agents (PTAs) that are sensitive to near-infrared (NIR) stimulation have been deemed as excellent materials candidate to produce transformable tissue engineering scaffolds via 4D printing [9]. On one hand, they offer the surgeons with practical convenience, as the printed scaffold can change its morphology by external force at the and maintain the deformed shape temporarily to pass through the implantation channel with a restricted volume. Afterwards, it recovers to its original shape and achieves adaptation at the implantation site through temperature changes induced by NIR stimulation [10]. On the other hand, mild hyperthermia triggered by NIR can effectively promote the proliferation of osteoblasts and angiogenic cells, which in turn promotes the formation of bone regeneration and vascularization [11].
4D 打印技术的出现为制作这种用于治疗不规则骨缺损的支架提供了一种有效的解决方案[5]。4D 打印的概念是在 打印的基础上增加一个时间维度,使打印支架的形状和形态在特定刺激下发生变化[6]。4D 打印最初源于形状记忆材料(SMMs)的 3D 打印。聚乳酸-共聚乙醇酸(PLGA)是一种可生物降解的形状记忆材料,被广泛应用于生物医学领域,并获得了美国食品及药物管理局(FDA)的批准[7]。PLGA 不仅具有良好的生物相容性,还可以通过改变乳酸和乙醇酸的比例来调节聚合物的玻璃化温度(Tg),使其接近人体可接受的温度[8]。近年来,温度响应型 SMM 与对近红外(NIR)刺激敏感的光热剂(PTAs)相结合,被认为是通过 4D 打印生产可转化组织工程支架的绝佳候选材料[9]。一方面,它们为外科医生提供了实际的便利,因为打印的支架可以在 ,通过外力改变其形态,并暂时保持变形的形状,以通过体积受限的植入通道。之后,通过近红外刺激引起的温度变化,它又会恢复到原来的形状,实现植入部位的适应性[10]。另一方面,近红外引发的轻度高热可有效促进成骨细胞和血管生成细胞的增殖,进而促进骨再生和血管形成[11]。
As a commonly used PTAs, nanoparticles possess excellent biocompatibility owning to its degradability in vivo [12], setting it apart from non-biodegradable PTAs such as graphene, molybdenum disulfide [13]. Meaningfully, nanoparticles not only possess marvelous photothermal effect, but also can promote osteogenesis of mesenchymal stem cells (MSCs) through magnetic response [14]. However, nanoparticles have limited hydrophilicity which reduces their biocompatibility. Therefore, surface modification on nanoparticles is needed.
作为一种常用的 PTA, 纳米粒子具有良好的生物相容性,可在体内降解[12],使其有别于石墨烯、二硫化钼等不可生物降解的 PTA [13]。有意义的是, 纳米粒子不仅具有神奇的光热效应,还能通过磁响应促进间充质干细胞(MSCs)的成骨过程[14]。然而, 纳米粒子的亲水性有限,降低了其生物相容性。因此,需要对 纳米粒子进行表面改性。
In this study, a dual-responsive bone tissue engineering scaffold was fabricated through 4D printing by incorporating magnetic nanoparticles into PLGA/HA composite matrices (designated as PHFS) and was applied to treat irregular bone defects. The scaffold subjected to NIR stimulation could be folded into a "slim" state and magnetically navigated to the defect site through a minimally invasive way with the assistant of external static magnetic field (SMF), and further recover to their initial shapes by the second NIR stimulation to adapt to the defect boundaries precisely. In vitro and in vivo studies showed that the magnetic effect of nanoparticles and their NIR-induced photothermal effect could jointly enhance the osteogenesis and angiogenesis by upregulating the gene expression of PI3K/AKT signaling pathway. Taken together, by combining the dual responsiveness of nanoparticles to NIR and SMF with on-demand shape changing/
本研究将 磁性纳米粒子融入 PLGA/HA 复合基质(命名为 PHFS),通过 4D 打印制作了一种双响应骨组织工程支架,并将其用于治疗不规则骨缺损。受近红外刺激的支架可折叠成 "纤细 "状态,在外部静磁场(SMF)的辅助下通过微创方式磁导航到缺损部位,并在第二次近红外刺激下进一步恢复到初始形状,以精确适应缺损边界。体外和体内研究表明, 纳米粒子的磁效应和近红外诱导的光热效应可以通过上调 PI3K/AKT 信号通路的基因表达,共同促进骨生成和血管生成。综上所述,通过将 纳米粒子对近红外和 SMF 的双重响应性与按需改变形状/形状的功能结合起来,可以有效地促进骨生成和血管生成。
Scheme 1. Schematic diagram of scaffold synthesis, its precise fitting to bone defects through Near-infrared (NIR) stimulation and SMF navigation, and its mechanism of osteogenesis and angiogenesis in the rat defect site under dual-response stimulation.
方案 1. 支架合成示意图、通过近红外(NIR)刺激和 SMF 导航与骨缺损精确贴合示意图,以及在双重反应刺激下大鼠缺损部位的成骨和血管生成机制。

recovery capability of scaffold matrices, 4D printed PHFS scaffold can be easily delivered to the irregular bone defect and effectively induce accelerated bone regeneration with improved vascularization.
利用支架基质的恢复能力,4D 打印 PHFS 支架可以很容易地输送到不规则的骨缺损处,并有效地诱导加速骨再生,同时改善血管化。

2. Results and discussion
2.结果和讨论

2.1. Design of PLGA/HA/Fe scaffold
2.1.PLGA/HA/Fe 支架的设计

To effectively treat critical irregular bone defects, a dual-response PLGA/HA/Fe (PHFS) scaffold was designed and printed and implanted into defects via a minimally invasive way. This multifunctional scaffold had a specific permanent shape to match the irregular bone defect at body temperature, it can be deformed by external force at to achieve minimally invasive implantation with the assistance of external magnetic force at room temperature. Once the deformed scaffold was moved to the defect site, the temperature of the scaffold can be remotely increased to via NIR to enable the recovery adaptation to the irregular defect boundary (Scheme 1b). Moreover, the synergistic stimulation of SMF and NIR effectively promoted both in vitro and in vivo osteogenesis and angiogenesis via the mild hyperthermia generated by nanoparticles in the PHFS scaffold, since the interaction of SMF with nanoparticles activated the signaling pathway and the photothermal effect generated by nanoparticles upregulated the high expression of heat shock protein (HSP90), which further activated the phosphorylation degree of AKT (Scheme 1c) [15]. Towards the function of each scaffold component, nanoparticles were used as the key agent to provide the scaffold with both photothermal and magnetic capabilities. Compared to pristine nanoparticles, nanoparticles coated with a thin layer of silica (i.e., nanoparticles) had much hydrophilicity and biocompatibility [16]. Hydroxyapatite (HA) nanoparticles were employed to provide the scaffold with desirable mechanical strength and osteoconductivity [17], while PLGA was used as the matrix to load nanoparticles and HA, and responsible for shaping of the scaffold.
为了有效治疗严重的不规则骨缺损,我们设计并打印了一种PLGA/HA/Fe (PHFS)双响应支架,并通过微创方式将其植入缺损处。这种多功能支架在体温下具有与不规则骨缺损相匹配的特定永久形状,它可以在 的外力作用下变形,在室温下借助外磁力实现微创植入。将变形支架移至缺损部位后,可通过近红外远程将支架温度升至 ,使其恢复适应不规则的缺损边界(方案 1b)。此外,通过 纳米粒子在 PHFS 支架中产生的温和高热,SMF 和近红外的协同刺激有效促进了体外和体内的成骨和血管生成、因为SMF与 纳米粒子的相互作用激活了 信号通路,而 纳米粒子产生的光热效应上调了热休克蛋白(HSP90)的高表达,从而进一步激活了AKT的磷酸化程度(方案1c)[15]。为了实现支架各组成部分的功能, 纳米粒子被用作使支架同时具有光热和磁性功能的关键介质。与原始的 纳米粒子相比,涂有一薄层二氧化硅的 纳米粒子(即 纳米粒子)具有更强的亲水性和生物相容性[16]。羟基磷灰石(HA)纳米颗粒用于为支架提供理想的机械强度和骨传导性[17],而聚乳酸乙二醇酯(PLGA)则用作负载 纳米颗粒和 HA 的基质,并负责支架的塑形。

2.2. Synthesis and Characterization of @SiO nanoparticles
2.2. @SiO 纳米粒子的合成与表征

Under the induction of the magnetic field, a chain structure composed of nanoparticles was successfully formed. Transmission electron microscope (TEM) images showed that the nanoparticles with an average diameter of were peripherally wrapped by a thin layer of silica (thickness: ) and were arranged in chains under a SMF (Fig. 1a). In order to analyze the principle of bonding of and silica, the zeta potentials of tetraethyl orthosilicate (TEOS), and were measured. The results showed that TEOS and were positive ) and negative ) respectively, while the synthesized had a potential of , as shown in Fig. 1b.
在磁场的诱导下,成功形成了由 纳米粒子组成的链状结构。透射电子显微镜(TEM)图像显示,平均直径为 纳米粒子外围被一薄层二氧化硅(厚度: )包裹,并在 SMF 下呈链状排列(图 1a)。为了分析 和二氧化硅的结合原理,测量了正硅酸四乙酯(TEOS)、 的 Zeta 电位。结果表明,TEOS 和 分别为正 ) 和负 ) ,而合成的 的电位为 ,如图 1b 所示。
In order to analyze the components of the synthesized nanoparticles, X-ray diffractometer (XRD) and fourier transform infra-red spectrometer (FTIR) tests were performed on , separately. Fig. 1c shows the XRD patterns of and @SiO 2 . The diffraction peaks of , and correspond to (220), (311), (400), (422), (511), and (440), respectively [18]. It can be seen that both samples have the characteristic peak of , which indicates that the modification does not cause significant changes in the crystal structure of the magnetic nanoparticles. A broad diffraction peak at around is due to the amorphous structure of the silica shell [19]. Fig. 1d displays the FTIR spectrums of and nanoparticles. In the curve of , the peak around represents the vibration of the [20]. Besides, the peak at represents the vibration of the and the peak at is typical for .
为了分析合成纳米粒子的成分,对 分别进行了 X 射线衍射仪(XRD)和傅立叶变换红外光谱仪(FTIR)测试。图 1c 显示了 @SiO 2 的 XRD 图样。 , 和 的衍射峰分别对应于(220)、(311)、(400)、(422)、(511)和(440)[18]。可以看出,两个样品都有 的特征峰,这表明改性并没有引起磁性纳米粒子晶体结构的显著变化。在 左右的宽衍射峰是由于二氧化硅外壳的无定形结构造成的[19]。图 1d 显示了 纳米粒子的傅立叶变换红外光谱。在 的曲线上, 附近的峰代表 的振动 [20]。此外, 处的峰代表 的振动,而 处的峰是 的典型峰。
The magnetic properties of and nanoparticles were examined by vibrating sample magnetometer (VSM). The VSM
纳米粒子的磁性能通过振动样品磁力计(VSM)进行了检测。VSM
Fig. 1. Characterization of and nanoparticles. a) TEM images of nanoparticles. b) Zeta potentials of and nanoparticles. c) XRD patterns of and nanoparticles. d) FTIR of and nanoparticles. e and f) Magnetization curves and magnetization behaviors at magnetic field from -200 to 200 Oe.
图 1. 纳米粒子的表征。a) 纳米粒子的 TEM 图像。b) 纳米粒子的 Zeta 电位。c) 纳米粒子的 XRD 图。d) 纳米粒子的傅立叶变换红外光谱。e 和 f) 磁化曲线以及在 -200 至 200 Oe 磁场下的磁化行为。

results showed that the synthesized nanoparticles possessed excellent magnetic properties. According to the result shown in Fig. 1e, the saturation magnetization of nanoparticles is 68.35 , while that of nanoparticles is . It is worth noting that the saturation magnetization of is lower than that of since the addition of reduces the weight ratio of in [22]. Besides, Fig. 1f shows that both magnetic powders possess acceptable magnetization behavior.
结果表明,合成的 纳米粒子具有优异的磁性能。根据图 1e 所示的结果, 纳米粒子的饱和磁化率为 68.35 ,而 纳米粒子的饱和磁化率为 。值得注意的是,由于 的加入降低了 中的重量比,因此 的饱和磁化率低于 [22]。此外,图 1f 显示两种磁性粉末都具有可接受的磁化特性。

2.3. Fabrication and Characterization of 4D near-infrared responsive magnetic scaffolds
2.3.四维近红外响应磁性支架的制作与表征

In order to investigate the thermal behavior of the modified print inks, four groups of print inks, PLGA group (designated as PLGA), PLGA/ (w/w of 7:3) group (designated as PH), PLGA/HA/Fe content of ) group (designated as PHF), and PLGA/HA/ content of 4 wt ) group (designated as ), were subjected to differential scanning calorimeter (DSC) tests and thermogravimetric analysis (TGA). The DSC test results (Fig. 2a) revealed that the addition of and increased the glass transition temperatures (Tg) of the scaffolds to and , respectively, as compared to PLGA ). The compressive strength and elastic modulus of scaffolds decreased significantly when heated to a temperature above its , which in turn allowed compression and deformation of the scaffold for easy implantation in the irregular bone defect. Hence, considering the stability of the scaffold architecture under human body temperature conditions and the fact that moderate thermal stimulus benefits to promote the bone regeneration, the scaffold is suitable for subsequent biological studies [13b,23]. The results of TGA (Fig. 2b) proved that all groups own superb decomposition temperatures ), which ensures the stability of scaffolds after
为了研究改性印刷油墨的热行为,对四组印刷油墨进行了差示扫描量热计(DSC)测试和热重分析(TGA)。这四组印刷油墨分别是:PLGA 组(命名为 PLGA)、PLGA/ (重量比为 7:3)组(命名为 PH)、PLGA/HA/Fe 含量为 )组(命名为 PHF)和 PLGA/HA/ 含量为 4 wt )组(命名为 )。DSC 测试结果(图 2a)显示,与 PLGA ) 相比,添加 后,支架的玻璃化转变温度(Tg)分别提高到 。当加热到高于 的温度时,支架的抗压强度和弹性模量明显降低,这反过来又使支架发生压缩和变形,便于植入不规则的骨缺损处。因此,考虑到支架结构在人体温度条件下的稳定性,以及适度的热刺激 对促进骨再生的益处, 支架适合后续的生物学研究[13b,23]。TGA 的结果(图 2b)证明,所有组都具有极好的分解温度 ),这保证了支架在温度升高后的稳定性。
Fig. 2. Characterization of PLGA, PH, PHF, PHF@SiO scaffolds. a) Glass transition temperature of PLGA, PH, PHF, . b) Thermal degradation behavior of PLGA, PH, PHF, PHF@SiO 2 . c) Water contact angle of PLGA, PH, PHF, PHF@SiO . d) The XRD patterns of PLGA, PH, PHF, PHF@SiO2. e) The FTIR spectra of PLGA,