2024_06_19_86a4bfc39e7b639451c4g

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 130021c 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：

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

的

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）、

和

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

和

@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

和

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

和

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.

和

纳米粒子的表征。a)

纳米粒子的 TEM 图像。b)

和

纳米粒子的 Zeta 电位。c)

和

纳米粒子的 XRD 图。d)

和

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

[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

在

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

和

后，支架的玻璃化转变温度（Tg）分别提高到

和

。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,

. f) The SEM images (25X, 50X, 100X, 200X) of PHF (top) and PHF@SiO

(bottom). g) The EDS spectroscopy of

scaffold.
图 2.

a) PLGA、PH、PHF、PHF@SiO

支架的玻璃化温度。 b) PLGA、PH、PHF、PHF@SiO 2 的热降解行为。 c) PLGA、PH、PHF、PHF@SiO

的水接触角。d) PLGA、PH、PHF、PHF@SiO2 的 XRD 图。 e) PLGA、

的傅立叶变换红外光谱。 f) PHF（上）和 PHF@SiO

（下）的 SEM 图像（25X、50X、100X、200X）。 g)

支架的 EDS 光谱。
implantation in the body. Hydrophilicity of materials is an essential parameter to tune the cell adhesion and extension, which is important to bone regeneration [24]. As shown in Fig. 2c, the water contact angle of the

scaffold was significantly lower compared to PHF scaffold, suggesting PHF@

scaffold had a suitable hydrophilicity for inducing bone regeneration. The XRD spectrums (Fig. 2d) of

scaffolds possess characteristic peaks of

and

, whereas PLGA has no obvious diffraction peaks due to its amorphous nature [25]. Similarly, the FTIR results of the

scaffolds show the presence of

, HA and

components, as shown in Fig. 2e. In order to observe the microscopic morphology and elemental distribution of the scaffolds, scanning electron microscopy (SEM) and energy-dispersive spectrometry (EDS) were performed on the PHF group and

group. The SEM results (Fig. 2f) showed that both PHF and PHF@SiO group scaffolds were porous and exhibited a hierarchical cross mesh structure. The surface roughness of the scaffolds was obvious. This rough surface favors cell growth and proliferation, while the porous structure facilitates nutrient exchange and micro-vessel generation [26]. For the PHF group and PHF@

group, the EDS results showed that the scaffold surface uniformly exhibited C, O, Ca, P and Fe elements. Among them,

and

elements were from PLGA,

and

elements were from

, and

elements were derived from

and

nanoparticles (Fig. S1). For PHF@SiO

group, the Si element was from

nanoparticles (Fig.

). This indicates that

and

nanoparticles were successfully incorporated into the scaffolds and uniformly distributed on the surface of the scaffolds.
植入体内。材料的亲水性是调节细胞粘附性和延伸性的重要参数，而细胞粘附性和延伸性对骨再生非常重要[24]。如图 2c 所示，与 PHF 支架相比，

支架的水接触角明显较低，这表明 PHF@

支架具有诱导骨再生的合适亲水性。

支架的 XRD 光谱（图 2d）具有

和

的特征峰，而 PLGA 由于其无定形性质没有明显的衍射峰[25]。同样，

支架的傅立叶变换红外光谱结果显示存在

、HA 和

成分，如图 2e 所示。为了观察支架的微观形态和元素分布，对 PHF 组和

组进行了扫描电子显微镜（SEM）和能量色散光谱（EDS）分析。扫描电子显微镜结果（图 2f）显示，PHF 组和 PHF@SiO 组支架均为多孔结构，并呈现分层交叉网状结构。支架表面粗糙度明显。这种粗糙的表面有利于细胞的生长和增殖，而多孔结构则有利于营养交换和微血管的生成[26]。对于 PHF 组和 PHF@

组，EDS 结果显示支架表面均匀地呈现出 C、O、Ca、P 和 Fe 元素。其中，

和

元素来自 PLGA，

和

元素来自

，

元素来自

和

纳米颗粒（图 S1）。在 PHF@SiO

组中，硅元素来自

纳米粒子（图

）。这表明

和

纳米颗粒成功地融入了支架并均匀地分布在支架表面。

Considering the hydrophilicity and deformation temperature of the scaffolds, the PHF@SiO 2 scaffold was selected for testing in the subsequent experiments, and the following groups were set up according to the different weight ratios of

(group

考虑到支架的亲水性和变形温度，在随后的实验中选择了 PHF@SiO 2 支架进行测试，并根据

的不同重量比设置了以下组别（组别

）、

Fig. 3. Characterization of PH, PHFS0.02, PHFS0.04, PHFS0.08 and PHFS0.16 scaffolds. a) Mechanical properties of PH, PHFS0.02, PHFSO.04, PHFS0.08, PHFS0.16 scaffolds. b) Weight remaining ration of PH, PHFS0.02, PHFS0.04, PHFS0.08, PHFS0.16 scaffolds during degradation for 8 weeks. c and d) Magnetization curves and magnetization behaviors of PH, PHFS0.02, PHFS0.04, PHFS0.08, PHFS0.16 scaffolds at magnetic field from -300 to 300 Oe. e and f) Photothermal images and thermal curves of PH, PHFS0.02, PHFS0.04, PHFS0.08, PHFS0.16 scaffolds at a power density of

. g) Shape recovery ratio and recovery time of PH, PHFS0.02, PHFS0.04, PHFS0.08, PHFS0.16 scaffolds in the water at

. h) Demonstration images of shape recovery of PHFS0.04 scaffolds. i) Demonstration of precisely and minimally invasive implantation by NIR heating and magnetic navigation. j) Recycling heating profile of PHFS0.04 scaffolds with an

laser irradiation (

) for three laser on/off cycles. k) Shape fixation ratio of

, PHFS0.04, PHFS0.08, PHFS0.16 scaffolds in the water at

.
图 3.a) PH、PHFS0.02、PHFS0.04、PHFS0.08 和 PHFS0.16 支架的机械性能、b) PH、PHFS0.02、PHFS0.04、PHFS0.08 和 PHFS0.16 支架在降解 8 周后的剩余重量比。08、PHFS0.16 支架在 -300 至 300 Oe 磁场下的磁化曲线和磁化行为。 e 和 f) PH、PHFS0.02、PHFS0.04、PHFS0.08、PHFS0.16 支架在

功率密度下的光热图像和热曲线。 g) PH、PHFS0.02、PHFS0.04、PHFS0.08、PHFS0.16 支架在水中的形状恢复率和恢复时间，

。 h) PHFS0.04 支架形状恢复的演示图像。 i) 通过近红外加热和磁导航进行精确微创植入的演示。j) PHFS0.04 支架在

激光照射下 (

) 三个激光开/关周期的循环加热曲线。 k)

、PHFS0.04、PHFS0.08、PHFS0.16 支架在水中的形状固定率，网址为

。
(group PHFS0.02),

(group PHFS0.04),

(group PHFS0.08), and

(group PHFS0.16). It can be seen from Fig. 3a that the addition of

increased the elastic modulus and compressive strength of the scaffolds compared to

group, and these values continuously increased with increasing

content. It can be attributed to the fact that rigid

particles can effectively resist the deformation of polymer chains under external forces [27]. As a biological bone substitution scaffold, the degradation rate of the scaffold should match the rate of bone regeneration. Compared to the

group, the addition of

particles did not significantly affect the degradation rate of PHFS groups. The scaffolds were immersed in phosphate buffered solution (PBS) and placed in a

incubator for 8 weeks to investigate the degradation behavior. The results (Fig. 3b) showed that the weight loss of

group was approximately

, while the other groups gradually slowed down the weight loss (24%-29 %) with the increase of

content. The results of VSM (Fig. 3c, d) indicated that the magnetic properties of the scaffolds increased with the increasing of magnetic nanoparticle contents. Besides, the magnetic attraction of scaffold stripe indicates that the scaffolds containing

components present superb magnetic properties (Fig. S2).
(组 PHFS0.02）、

（组 PHFS0.04）、

（组 PHFS0.08）和

（组 PHFS0.16）。从图 3a 可以看出，与

组相比，添加

增加了支架的弹性模量和抗压强度，并且这些值随着

含量的增加而持续增加。这可能是由于刚性

颗粒能有效抵抗聚合物链在外力作用下的变形[27]。作为一种生物骨替代支架，支架的降解率应与骨再生率相匹配。与

组相比，添加

颗粒对 PHFS 组的降解率没有明显影响。将支架浸入磷酸盐缓冲溶液（PBS）中，并在

培养箱中放置 8 周，以研究其降解行为。结果（图 3b）显示，

组的失重率约为

，

，而其他组随着

含量的增加，失重率逐渐减慢（24%-29%）。VSM 结果（图 3c、d）表明，支架的磁性能随着磁性纳米粒子含量的增加而增强。此外，支架条纹的磁吸引力表明，含有

成分的支架具有极佳的磁性能（图 S2）。

The photothermal conversion efficiency of SMMs affects the deformation behavior of the printed scaffold as well as the thermal stimulation effect on cells. The PHFS0.04 group was firstly selected to observe the NIR thermal curves under different power densities. The results displayed that the power intensity can determine the final stabilization temperature of scaffolds (Fig. S3). Then, the thermal curves of different scaffolds were observed at a power density of

(Fig. 3e, f). Different group scaffolds showed comparable thermal curves at the same power density. Considering that the

of the

scaffolds was

, the power density of the NIR light source was set as

in the subsequent experiments. To assess the photothermal stability of the scaffolds, PHFS0.04 scaffolds underwent recycling heating tests, demonstrating that the PHFS0.04 scaffold exhibited exceptional photothermal stability (Fig. 3j). In addition, it can be seen from Fig. 3g that the deformation recovery of scaffolds in different groups was not significantly affected when a small content of

nanoparticles was incorporated. Besides the shape recovery ratio, the shape fixation ratio of the scaffold is also critical for its practical application. The results in Fig. 3k showed that all groups of the scaffolds had excellent shape fixation ratios. The Fig. 3h illustrated the deformation process of the

scaffolds. As a thermally activated shape memory material, the

scaffold exhibits a structure with randomly oriented molecular chains and high entropy values at ambient temperature. The

scaffolds can be stretched under external forces when heated above their

, leading to a decrease in the entropy value of the system. Cooling the scaffold under tension causes the molecular chains to become less mobile and remain elongated, effectively trapping the induced internal stress as elastic potential energy within the molecular framework. Subsequent reheating of the scaffold above its Tg allows the stored elastic potential energy to realign the molecular chains, thus driving the material back to its predefined original shape. The responsiveness of the

nanoparticles to external magnetic fields determines the success of magnetic navigation. To verify the feasibility of magnetic navigation-aided implantation, a magnetic stripe was used to attract the PHF@

scaffolds and guide its movement. The results showed that the PHF@

scaffolds could realize fast and precise position adjustment under the action of SMF (Video S1). To verify the feasibility of minimally invasive scaffold implantation, the initial shapes of personalized printed scaffolds were firstly altered by external force at

which was above the glass transition temperature. After cooling, the temporary shape was fixed and the precisely implantation of the deformed scaffolds via thin catheter was successfully realized with the assistance of a static magnetic field. Finally, the scaffolds were heated by NIR irradiation and get recovered to their original shape and adapted to the defect boundaries (Fig. 3i).
SMM 的光热转换效率会影响印刷支架的变形行为以及对细胞的热刺激作用。首先选择 PHFS0.04 组观察不同功率密度下的近红外热曲线。结果表明，功率密度可以决定支架的最终稳定温度（图 S3）。然后，观察了

功率密度下不同支架的热曲线（图 3e，f）。在相同的功率密度下，不同组的支架显示出相似的热曲线。考虑到

支架的

为

，因此在随后的实验中将近红外光源的功率密度设定为

。为了评估支架的光热稳定性，PHFS0.04 支架进行了循环加热试验，结果表明 PHFS0.04 支架具有优异的光热稳定性（图 3j）。此外，从图 3g 中还可以看出，当加入少量

纳米粒子时，不同组别支架的变形恢复没有受到明显影响。除了形状恢复率，支架的形状固定率也是其实际应用的关键。图 3k 中的结果显示，各组支架的形状固定率都很好。图 3h 展示了

支架的变形过程。作为一种热激活形状记忆材料，

支架在常温下具有随机取向的分子链结构和较高的熵值。当

支架受热超过其

时，可在外力作用下拉伸，从而导致系统熵值降低。在张力作用下冷却支架会降低分子链的移动性并保持伸长，从而有效地将诱导的内应力作为弹性势能困在分子框架内。随后将支架重新加热至 Tg 以上，可使储存的弹性势能重新排列分子链，从而使材料恢复到预定的原始形状。

纳米粒子对外部磁场的反应能力决定了磁导航的成败。为了验证磁导航辅助植入的可行性，我们使用磁条吸引 PHF@

支架并引导其移动。结果表明，在SMF的作用下，PHF@

支架可以实现快速、精确的位置调整（视频S1）。为了验证微创支架植入的可行性，首先在高于玻璃转化温度的

，通过外力改变个性化打印支架的初始形状。冷却后，固定临时形状，并在静态磁场的辅助下通过细导管成功实现了变形支架的精确植入。最后，通过近红外照射加热支架，支架恢复到原来的形状并与缺陷边界相适应（图 3i）。

2.4. In vitro biocompatibility
2.4.体外生物相容性

Excellent biocompatibility is always a basic requirement for designing a new bone tissue engineering scaffold. To analyze the effect of

nanoparticles concentration on cells, cell viability on the scaffolds was assessed using a live/dead staining kit. The result of cell viability assay showed that PHFSO.04 groups possessed excellent biocompatibility (Fig. 4a, b). A Cell Counting Kit-8 (CCK-8) was used to assess the proliferation of rBMSCs on the scaffolds. From 3 day and 7 day results, the

nanoparticle concentrations of PHFSO.02 and PHFSO. 04 groups were suitable for cell proliferation (Fig. 4c). Besides, cytoskeletal staining results showed that cells were well stretched on PHFS0.04 scaffold (Fig. S4) and the results of three-dimensional cell viability assay and cytolytic hemolysis assay showed that the PHSF0.04 scaffold possessed excellent biocompatibility (Figs. S5 and S6). Therefore, from the perspective of biocompatibility, the PHFS0.04 group was selected for the subsequent experiments. Considering the stimulating effects of static magnetic fields (

) and NIR

) on the scaffolds, the subsequent experiments were grouped as follows: PLGA/HA (group

), PLGA/HA/4 w/w% Fe

(group PHFS), PLGA/HA/4 w/w

(group PHFS + SMF), PLGA/HA/4 w/w%

NIR (group PHFS + NIR), PLGA/HA/4 w/w% Fe

(group PHFS + NIR + SMF). In addition, the static magnetic field strength was set at

for this study because the magnetic field strength of

is favorable for the proliferation of rBMSCs [14b,28]. A live/dead staining and a Cell Counting Kit-8 (CCK8) assay were used to assess the proliferative effects and cell viability of NIR and SMF stimulation on rBMSCs. The live-dead results showed that the stimulation with SMF and NIR did not increase the percentage of dead cells (Fig. 4d, e). The CCK-8 results, as shown in Fig. 4f, revealed that the static magnetic field and NIR could effectively promote cell proliferation, and there was a superimposed effect of the two stimuli. To further affirm the biocompatibility of the scaffolds, the CCK-8 assay was used on HUVECs. The results indicated that the combination of SMF and NIR could enhance the proliferation of HUVECs (Fig. S7).
优良的生物相容性一直是设计新型骨组织工程支架的基本要求。为了分析

纳米粒子浓度对细胞的影响，使用活/死染色试剂盒评估了支架上的细胞活力。细胞存活率检测结果表明，PHFSO.04 组具有良好的生物相容性（图 4a、b）。细胞计数试剂盒-8（CCK-8）用于评估支架上 rBMSCs 的增殖情况。从 3 天和 7 天的结果来看，PHFSO.02 和 PHFSO.04 组的

纳米粒子浓度适合细胞增殖。04 组适合细胞增殖（图 4c）。此外，细胞骨架染色结果表明，细胞在 PHFS0.04 支架上得到了很好的拉伸（图 S4），三维细胞活力检测和细胞溶血检测结果表明 PHSF0.04 支架具有良好的生物相容性（图 S5 和 S6）。因此，从生物相容性的角度考虑，后续实验选择了 PHFS0.04 组。考虑到静态磁场 (

) 和近红外

) 对支架的刺激作用，后续实验分组如下：PLGA/HA（组

），PLGA/HA/4 w/w% Fe

（组 PHFS），PLGA/HA/4 w/w

（组 PHFS + SMF），PLGA/HA/4 w/w%

NIR（组 PHFS + NIR），PLGA/HA/4 w/w% Fe

（组 PHFS + NIR + SMF）。此外，由于

的磁场强度有利于 rBMSCs 的增殖 [14b,28]，因此本研究将静态磁场强度设为

。活体/死体染色和细胞计数试剂盒-8（CCK8）检测被用来评估近红外和SMF刺激对rBMSCs的增殖效应和细胞活力。活死细胞染色结果显示，SMF 和近红外刺激并没有增加死细胞的比例（图 4d、e）。如图 4f 所示，CCK-8 结果显示，静磁场和近红外能有效促进细胞增殖，两种刺激存在叠加效应。为了进一步确定支架的生物相容性，我们对 HUVECs 进行了 CCK-8 试验。结果表明，SMF 和近红外的组合能增强 HUVEC 的增殖（图 S7）。

2.5. In vitro osteogenesis evoked by SMF and NIR
2.5.SMF 和近红外诱发的体外骨生成

Osteoblast-induced biomineralization is important in the repair of bone defects. To assess the osteogenic activation ability of the scaffolds in response to NIR and SMF, quantitative and qualitative alkaline phosphatase (ALP) experiments were performed on the scaffolds. The results of 7 and 14 days (Fig. 5a, b) showed that

nanoparticles, NIR stimulation and SMF stimulation increased the expression of ALP compared with the blank and PH groups. Besides, the group with magnetic stimulation was slightly more effective than NIR stimulation group. The reason may be due to the fact that the stimulation time of magnetic field was significantly longer than that of NIR thermal stimulation. The 28 -day alizarin red (AR) staining results also indicated that the stimulus-responsive groups of SMF and NIR improved the biomineralization capacity by increasing the mineralization level of cellular mineralized nodules, as shown in Fig. 5a. In gene level, four genes were involved to assess the osteogenesis capacity, including COL-1, OCN, RUNX2 and BMP-2. RUNX2 is usually regarded as one of the genes expressed in early osteogenesis, whereas OCN and COL-1 are genes expressed in late osteogenesis [29]. After co-culturing the scaffolds with osteogenic-induced rBMSCs for 7 and 14 days, real-time quantitative polymerase chain reaction (RT-qPCR) was applied to evaluate osteogenic-related gene expression. The results showed that all osteogenesis-related genes (COL-1, OCN, RUNX2 and BMP-2) were significantly upregulated in the PHFS + NIR + SMF group (Fig. 5c). For the RUNX2 gene, the degree of expression was more pronounced at 7 days than at 14 days, and the stimulation effect of NIR was higher than that of SMF. However, for the expression of COL-1, OCN and BMP-2, the continuous stimulating effect of a static magnetic field was more effective than intermittent thermal stimulation. To further verify the
成骨细胞诱导的生物矿化对骨缺损的修复非常重要。为了评估支架在近红外和 SMF 作用下的成骨活化能力，对支架进行了碱性磷酸酶（ALP）的定量和定性实验。7 天和 14 天的实验结果（图 5a、b）表明，与空白组和 PH 组相比，

纳米粒子、近红外刺激和 SMF 刺激均增加了 ALP 的表达。此外，磁刺激组的效果略高于近红外刺激组。原因可能是磁场刺激的时间明显长于近红外热刺激。28 天的茜素红（AR）染色结果也表明，SMF 和 NIR 刺激反应组通过提高细胞矿化结核的矿化水平，提高了生物矿化能力，如图 5a 所示。在基因水平上，有四个基因参与了成骨能力的评估，包括 COL-1、OCN、RUNX2 和 BMP-2。RUNX2通常被认为是成骨早期表达的基因之一，而OCN和COL-1则是成骨晚期表达的基因[29]。将支架与成骨诱导的 rBMSCs 共同培养 7 天和 14 天后，应用实时定量聚合酶链反应（RT-qPCR）评估成骨相关基因的表达。结果显示，所有成骨相关基因（COL-1、OCN、RUNX2 和 BMP-2）在 PHFS + NIR + SMF 组中都显著上调（图 5c）。就 RUNX2 基因而言，7 天时的表达程度比 14 天时更明显，而且近红外的刺激效果高于 SMF。然而，对于 COL-1、OCN 和 BMP-2 基因的表达，静态磁场的持续刺激作用比间歇性热刺激更有效。为了进一步验证

Fig. 4. The biocompatibility assay of the scaffolds. a and b) The cell viability assay of PH, PHFS0.02, PHFS0.04, PHFS0.08 and PHFS0.16 scaffolds. c) The CCK-8 assay of Blank, PH, PHFS0.02, PHFS0.04, PHFS0.08 and PHFS0.16 scaffolds.

and e) The cell viability assay of PH, PHFS, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups. f) The CCK-8 assay of Blank, PH, PHFS, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups.
图 4.a 和 b) PH、PHFS0.02、PHFS0.04、PHFS0.08 和 PHFS0.16 组支架的细胞存活率检测。c) 空白组、PH、PHFS0.02、PHFS0.04、PHFS0.08 和 PHFS0.16 组支架的 CCK-8 检测。

和 e) PH、PHFS、PHFS + SMF、PHFS + NIR 和 PHFS + NIR + SMF 组的细胞活力检测。 f) 空白、PH、PHFS、PHFS + SMF、PHFS + NIR 和 PHFS + NIR + SMF 组的 CCK-8 检测。

biomineralization activation effect of PHFS + NIR + SMF at the protein level, rBMSCs co-cultured with the scaffolds were stained for protein Immunofluorescence Staining. The results (Fig. 5d, e) showed that magnetic and thermal stimulation of PHFS scaffolds promoted the expression of osteogenesis-related proteins (ALP, COL-1, OCN and RUNX2) in rBMSCs, and the synergistic effect of dual stimulation was superior to thermal or magnetic stimulation alone.
为了从蛋白质水平研究 PHFS + NIR + SMF 的生物矿化激活效应，对与支架共培养的 rBMSCs 进行了蛋白质免疫荧光染色。结果（图 5d、e）表明，PHFS 支架的磁刺激和热刺激促进了成骨相关蛋白（ALP、COL-1、OCN 和 RUNX2）在 rBMSCs 中的表达，而且双重刺激的协同效应优于单独的热刺激或磁刺激。

2.6. Transcriptomics analysis and western blotting (WB) assay of osteogenesis
2.6.成骨的转录组学分析和西部印迹（WB）检测

To explore the possible molecular mechanisms by which SMF and NIR stimulation promote osteogenesis, we performed transcriptomic analysis on the Blank control, PHFS + SMF and PHFS + SMF + NIR groups. Firstly, the FPKM results confirmed the reliability of the data from the three samples (Fig. S8). The closer the correlation coefficient is to 1 , the indicates the higher the similarity of expression patterns between samples. Then, differential gene statistics were used to determine changes in differential genes between groups, as shown in Fig. 6a. The results showed that there were 1,284 differential genes between the PHFS + SMF + NIR group and the blank group, including 884 upregulated genes and 400 down-regulated genes. Meanwhile, there were 310 differential genes between the PHFS + SMF + NIR and PHFS + SMF groups, including 54 up-regulated and 256 down-regulated genes (Fig. S9). Besides, the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that the PHFS + SMF + NIR group significantly activated the PI3K/AKT signaling pathway (Fig. 6b). Furthermore, the results of the differential gene volcano plot indicated that the PHFS + SMF + NIR group up-regulated the expression of HSP90 compared to the PHFS + SMF group (Fig. 6c). Herein, according to the results of the volcano plot, it was demonstrated that NIR thermal stimulation upregulated the high expression of the HSP90 gene. To further investigate the osteogenic induction of rBMSCs by magnetic field and NIR at the protein level, the blank control, PHFS + SMF, PHFS + NIR, PHFS + NIR + SMF groups were selected for WB experiments. The WB results showed that pPI3K and pAKT were highly expressed in the PHFS + NIR + SMF group (Fig. 6d, e). Meanwhile, HSP90 protein showed high expression in PHFS + NIR and PHFS + NIR + SMF. Furthermore, the PHFS + NIR + SMF group also showed high expression of osteogenesisrelated proteins (COL-1, RUNX2 and OCN proteins), as shown in Fig.

and Fig.

. Therefore, based on the results of WB, we introduced that SMF stimulation was able to induce high expression of the PI3K/AKT signaling pathway. Meanwhile, NIR thermal stimulation further activated the phosphorylation of AKT by activating the high expression of HSP90 protein, which ultimately induces the high expression of downstream osteogenesis-related proteins (COL-1, RUNX2 and OCN proteins).
为了探索SMF和近红外刺激促进成骨的可能分子机制，我们对空白对照组、PHFS+SMF组和PHFS+SMF+近红外组进行了转录组学分析。首先，FPKM 结果证实了三个样本数据的可靠性（图 S8）。相关系数越接近 1，表明样本间表达模式的相似性越高。然后，如图 6a 所示，使用差异基因统计来确定组间差异基因的变化。结果显示，PHFS + SMF + NIR 组与空白组之间有 1284 个差异基因，其中上调基因 884 个，下调基因 400 个。同时，PHFS + SMF + NIR 组与 PHFS + SMF 组之间有 310 个差异基因，包括 54 个上调基因和 256 个下调基因（图 S9）。此外，京都基因组百科全书（KEGG）富集结果显示，PHFS + SMF + NIR 组显著激活了 PI3K/AKT 信号通路（图 6b）。此外，差异基因火山图的结果表明，与 PHFS + SMF 组相比，PHFS + SMF + NIR 组上调了 HSP90 的表达（图 6c）。由此可见，根据火山图的结果，近红外热刺激上调了 HSP90 基因的高表达。为了进一步研究磁场和近红外在蛋白质水平上对 rBMSCs 的成骨诱导作用，实验组分别选择空白对照组、PHFS + SMF 组、PHFS + NIR 组、PHFS + NIR + SMF 组进行 WB 实验。WB 结果显示，pPI3K 和 pAKT 在 PHFS + NIR + SMF 组中高表达（图 6d，e）。同时，HSP90蛋白在PHFS + NIR组和PHFS + NIR + SMF组均有高表达。此外，PHFS + NIR + SMF 组的成骨相关蛋白（COL-1、RUNX2 和 OCN 蛋白）也有高表达，如图

和图

所示。因此，根据 WB 结果，我们推测 SMF 刺激能够诱导 PI3K/AKT 信号通路的高表达。同时，近红外热刺激通过激活HSP90蛋白的高表达，进一步激活了AKT的磷酸化，最终诱导了下游成骨相关蛋白（COL-1、RUNX2和OCN蛋白）的高表达。

2.7. In vitro angiogenesis evoked by SMF and NIR
2.7.SMF 和近红外诱发的体外血管生成

For tissue-engineered scaffolds, effective vascularization can improve the bone regeneration ability of scaffold materials [30]. The vascularization of the scaffold materials not only provides adequate nutrition for migrating cells, but also facilitates oxygen exchange and product metabolism in osteoblasts [31]. Several studies indicated that magnetic or thermal stimulation of scaffolds can effectively activate the PI3K/AKT pathway and thus promote vascularization [11a,32]. To explore the activation of vascularization by SMF and NIR, wound healing experiments and transwell experiments were tested on

, PHFS, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups, respectively. For wound healing experiments, the results showed that the PHFS + NIR + SMF group had the best cell migration, and the wound was basically completely healed at

. The PHFS + SMF group migrated slightly faster than the PHFS + NIR group (Fig. 7a, b). The transwell assay also indicated that PHFS + NIR + SMF group possessed excellent migration capability compared with other groups (Fig. 7c, d). In addition, the results of tube formation assay displayed that a static magnetic field stimulation applied to the PHFS scaffold significantly promoted tube formation in HUVECs (Fig. 7e, f).
对于组织工程支架而言，有效的血管化可以提高支架材料的骨再生能力[30]。支架材料的血管化不仅能为迁移细胞提供充足的营养，还能促进成骨细胞的氧气交换和产物代谢[31]。多项研究表明，磁刺激或热刺激支架可有效激活 PI3K/AKT 通路，从而促进血管化[11a,32]。为探讨 SMF 和近红外对血管生成的激活作用，分别在

、PHFS、PHFS + SMF、PHFS + 近红外和 PHFS + 近红外 + SMF 组进行了伤口愈合实验和转孔实验。伤口愈合实验结果表明，PHFS + NIR + SMF 组的细胞迁移效果最好，在

，伤口基本完全愈合。PHFS + SMF 组的迁移速度略快于 PHFS + NIR 组（图 7a，b）。经孔试验也表明，与其他组相比，PHFS + NIR + SMF 组具有出色的迁移能力（图 7c，d）。此外，试管形成试验结果表明，对 PHFS 支架施加静态磁场刺激可显著促进 HUVECs 中试管的形成（图 7e，f）。

To further explore the ability of the PHFS + NIR + SMF group to induce angiogenesis, the vascularization-related gene expressions of HUVECs co-cultured with different groups (Blank, PH, PHFS, PHFS + SMF, PHFS + NIR, PHFS + NIR + SMF) were analyzed at the gene level. VEGF, PDGF, HIF-

, and

-SMA were selected for RT-qPCR experiments.
为了进一步探讨 PHFS + NIR + SMF 组诱导血管生成的能力，我们在基因水平上分析了与不同组（空白、PH、PHFS、PHFS + SMF、PHFS + NIR、PHFS + NIR + SMF）共培养的 HUVECs 的血管生成相关基因表达。选择 VEGF、PDGF、HIF-

和

-SMA 进行 RT-qPCR 实验。

Fig. 5. The Osteogenesis assay of PH, PHFS, PHFS + SMF, PHFS + NIR, PHFS + NIR + SMF groups. a) The qualitative assay of alkaline phosphatase (ALP, 7 and 14 days) and alizarin red (AR, 28 days). b) The quantitative assay of alkaline phosphatase (ALP, 7 and 14 days). c) The osteogenesis-related gene expressions of COL-1, OCN, RUNX2 and BMP-2 at 7 and 14 days. d) the protein Immunofluorescence Staining of ALP, COL-1, OCN and RUNX2 at 7 days. e) The corresponding semiquantitative analysis of the protein Immunofluorescence Staining of ALP, COL-1, OCN and RUNX2 at 7 days.
图 5.a) 碱性磷酸酶（ALP，7 天和 14 天）和茜素红（AR，28 天）的定性检测。c) 7 天和 14 天时 COL-1、OCN、RUNX2 和 BMP-2 的成骨相关基因表达。 d) 7 天时 ALP、COL-1、OCN 和 RUNX2 的蛋白质免疫荧光染色。

Fig. 6. Transcriptomics analysis and western blotting of the scaffolds. a) The differential gene statistics of Blank control, PHFS + SMF and PHFS + NIR + SMF groups. b) The Kyoto Encyclopedia of Genes and Genomes enrichment of Blank control and PHFS + SMF groups. c) Differential gene volcano plot of PHFS + SMF and PHFS + NIR + SMF groups. d) The signaling pathway of western blot of Blank, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups. e) The corresponding quantitative analysis of signaling pathway. f) The osteogenesis-related proteins of western blot of Blank, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups. g) The corresponding quantitative analysis of osteogenesis-related proteins.
图 6.a) Blank 对照组、PHFS + SMF 组和 PHFS + NIR + SMF 组的差异基因统计图。 b) Blank 对照组和 PHFS + SMF 组的京都基因组百科全书富集图。d) 空白对照组、PHFS + SMF 组、PHFS + NIR 组和 PHFS + NIR + SMF 组的 Western 印迹信号通路。 f) 空白对照组、PHFS + SMF 组、PHFS + NIR 组和 PHFS + NIR + SMF 组的 Western 印迹成骨相关蛋白。

Fig. 7. The angiogenesis assay of the scaffolds. a and b) The wound healing assay of PH, PHFS, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups. c and d) The transwell assay of PH, PHFS, PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups. e and f) The tube formation assay of PH, PHFS and PHFS + SMF groups.
图 7.a 和 b) PH 组、PHFS 组、PHFS + SMF 组、PHFS + NIR 组和 PHFS + NIR + SMF 组的伤口愈合试验；c 和 d) PH 组、PHFS 组、PHFS + SMF 组、PHFS + NIR 组和 PHFS + NIR + SMF 组的透孔试验；e 和 f) PH 组、PHFS 组和 PHFS + SMF 组的血管形成试验。

Compared with the blank control group, the gene expression of VEGF, PDGF, HIF-

, and

-SMA were significantly elevated in the PHFS + NIR + SMF group (Fig. 8a). For VEGF and PDGF genes, the vascularization of HUVEC was significantly increased in both the magnetic field stimulation group (PHFS + SMF) and the near-infrared thermal stimulation group (PHFS + NIR), and both stimuli showed a similar ability to induce angiogenesis. Meanwhile, the induction of HIF-

gene by magnetic fields was more pronounced whereas NIR was more effective in activating

-SMA gene. Protein immunofluorescence staining was selected to further assess the vascularization activation ability of PHFS + NIR + SMF group. The results showed significantly elevated expression of the proteins in HUVEC co-cultured with the PHFS + NIR + SMF group. The specific results showed that the dual-responsive PHFS + NIR + SMF group significantly upraised the expression of the VEGF, HIF-

and CD31 in HUVECs (Fig. 8b, f). To further verify the pathways activated by HUVECs angiogenesis, groups PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF were selected for western blot experiments. The results, as shown in Fig. 8c, d and e, showed that SMF and NIR stimulation
与空白对照组相比，VEGF、PDGF、HIF-

和

-SMA 的基因表达在 PHFS + NIR + SMF 组明显升高（图 8a）。就 VEGF 和 PDGF 基因而言，磁场刺激组（PHFS + SMF）和近红外热刺激组（PHFS + NIR）中 HUVEC 的血管生成都明显增加，两种刺激都显示出相似的诱导血管生成的能力。同时，磁场对 HIF-

基因的诱导作用更明显，而近红外对

-SMA 基因的激活作用更有效。为了进一步评估 PHFS + NIR + SMF 组的血管生成激活能力，我们选择了蛋白免疫荧光染色法。结果显示，与 PHFS + NIR + SMF 组共同培养的 HUVEC 蛋白表达明显升高。具体结果显示，双反应 PHFS + NIR + SMF 组显著提高了 HUVEC 中 VEGF、HIF-

和 CD31 的表达（图 8b，f）。为了进一步验证 HUVECs 血管生成的激活途径，我们选择 PHFS + SMF 组、PHFS + NIR 组和 PHFS + NIR + SMF 组进行了 Western 印迹实验。如图 8c、d 和 e 所示，结果表明 SMF 和 NIR 刺激

Fig. 8. The angiogenic capacity assay of the scaffolds. a) The angiogenesis -related gene expressions of VEGF, PDGF, HIF-

, and

-SMA at 3 days. b) the protein Immunofluorescence Staining of VEGF, HIF-

and CD31 at 3 days. c) The western blot analyses of

-actin, AKT, pAKT, and VEGF proteins. d and e) The quantitative assay of western blot. f) The corresponding semi-quantitative analysis of the protein Immunofluorescence Staining of VEGF, HIF-

and CD31 at 3 days.
图 8.a) 3 天后血管生成相关基因 VEGF、PDGF、HIF-

和

-SMA 的表达。b) 3 天后 VEGF、HIF-

和 CD31 的蛋白免疫荧光染色。c)

-actin、AKT、pAKT 和 VEGF 蛋白的 Western 印迹分析。 d 和 e) Western 印迹的定量分析。 f) 3 天后 VEGF、HIF-

和 CD31 蛋白免疫荧光染色的相应半定量分析。
effectively upregulated the phosphorylation level of AKT, which in turn promoted the expression of angiogenesis-related proteins (VEGF).
有效地上调了 AKT 的磷酸化水平，进而促进了血管生成相关蛋白（VEGF）的表达。

2.8. In vivo bone regeneration
2.8.体内骨再生

The results of in vitro osteogenesis and angiogenesis related experiments indicated that applying a static magnetic field and NIR stimulation to PHFS scaffolds can effectively promote biomineralization and vascularization. To further explore the bone regeneration ability of the PHFS + NIR + SMF group in vivo, a rat model of a critical defect in the skull was used for animal experiments. Briefly, a drill (

diameter) was used to create two

diameter circular critical bone defects in the center of the bilateral parietal bones, and then different groups of scaffolds were implanted into the defect sites. Considering that in the above in vitro experiments, both osteogenic and angiogenic capacities of the

group were lower than those of the other groups, the animal experiments were divided into the following groups: Blank control group, PHFS group, PHFS + SMF group, PHFS + NIR group, PHFS + NIR + SMF group. Blank and PHFS + NIR groups were used to measure the thermal curves of NIR stimulation in rats. The results displayed that the group containing the scaffolds reached a stabilized temperature (approximately

) under near-infrared light irradiation with

for

, while the blank control group maintained the normal body temperature of the rats (Fig. 9a, b). A schematic of a static magnetic field applied to a rat is shown in Fig. 9c. For achieving minimally invasive implantation, after the deformed scaffold was conveniently implanted into the cranial defect site of rats, the shape of the scaffold was recovered and precisely adapted to the border of the bone defect boundaries under the NIR stimulation (

), shown in Fig. 9d. To investigate the precise navigation function of static magnetic field during the scaffold implantation, a thin plate was placed over the rat cranial irregularity model to simulate a narrow implantation channel, and the static magnetic field was used to guide the scaffolds to be precisely implanted into the bone defect (Video S2), and then the scaffolds was deformed by NIR irradiation (

) to adapt to the irregular defect (Videos S3 and S4). The micro-CT results at 4 and 8 weeks showed that the bone defects in rats with stimulation applied recovered better than those in the group without stimulation, and the quality of bone regeneration in the PHFS + NIR + SMF group was superior to that of the PHFS + SMF or PHFS + NIR groups alone (Fig. 9f). Quantitative results showed that the bone volume fraction (BV/TV) in the PHFS + NIR + SMF group reached

and

at 4 and 8 weeks, respectively; and the bone surface (BS) reached

and

(Fig. 9e). In addition, the trabecular number (Tb.N) and trabecula thickness (Tb.Th) of PHFS + NIR + SMF group were also significantly increased compared with the blank group.
体外骨生成和血管生成相关实验的结果表明，对 PHFS 支架施加静态磁场和近红外刺激可有效促进生物矿化和血管生成。为了进一步探讨 PHFS + NIR + SMF 组的体内骨再生能力，我们使用颅骨严重缺损的大鼠模型进行动物实验。简单地说，用钻头（直径

）在双侧顶骨中央造成两个直径为

的圆形临界骨缺损，然后将不同组别的支架植入缺损部位。考虑到在上述体外实验中，

组的成骨能力和血管生成能力均低于其他组，因此将动物实验分为以下几组：空白对照组、PHFS 组、PHFS + SMF 组、PHFS + NIR 组、PHFS + NIR + SMF 组。空白对照组和 PHFS + 近红外组用于测量近红外刺激大鼠的热曲线。结果显示，含有支架的组在

的近红外线照射下达到稳定温度（约

），

，而空白对照组则保持大鼠的正常体温（图 9a、b）。对大鼠施加静态磁场的示意图见图 9c。为实现微创植入，将变形支架方便地植入大鼠颅骨缺损部位后，在近红外刺激下（

），支架的形状恢复并精确地适应骨缺损边界的边缘，如图 9d 所示。为了研究静态磁场在支架植入过程中的精确导航功能，在大鼠颅骨不规则模型上放置一块薄板模拟狭窄的植入通道，利用静态磁场引导支架精确植入骨缺损（视频 S2），然后通过近红外照射(

) 使支架变形以适应不规则缺损（视频 S3 和 S4）。4 周和 8 周时的显微 CT 结果显示，应用刺激的大鼠骨缺损恢复情况优于未应用刺激的大鼠，PHFS + NIR + SMF 组的骨再生质量优于 PHFS + SMF 或 PHFS + NIR 组（图 9f）。定量结果显示，PHFS + NIR + SMF 组的骨体积分数（BV/TV）分别在 4 周和 8 周达到

和

；骨表面（BS）分别达到

和

（图 9e）。此外，骨小梁数量（Tb.N）和骨小梁厚度（Tb.与空白组相比，PHFS + NIR + SMF 组的 Th 值也明显增加。

Furthermore, Hematoxylin-Eosin staining (H&E) and Masson trichrome staining at 4 weeks and 8 weeks were used to evaluate the bone regeneration. The

results indicated that there was a significant upturn in new bone formation in the PHFS + NIR + SMF group compared to the blank control group at 4 and 8 weeks (Fig. 10a). Similarly, the immunofluorescence staining for osteogenesis-related proteins showed high expression of ALP, COL-1 and OCN in the PHFS + NIR + SMF group compared with the blank control group (Fig. 10b, d). In Masson staining experiment, The PHFS + SMF, PHFS + NIR and PHFS + NIR + SMF groups showed more collagen formation than the blank control and PHFS groups. Besides, the results of Masson staining showed that the stimulation of NIR heating produced more microvessels (Fig. 10a). The next immunofluorescence staining for angiogenesis -related proteins confirmed this discovery. Protein expression of VEGF, CD31 and HIF-

was significantly raised in the PHFS + NIR and PHFS + NIR + SMF groups (Fig. 10c, e). In addition, 8-week animal sections showed that the rate of degradation of the implanted scaffolds in vivo matched the rate of new bone formation. To explore the toxicity of the implanted scaffolds in each group, the hearts, livers, spleens, lungs, and kidneys of rats at 4 weeks post-surgery were observed by H&E staining.
此外，4周和8周时的血红素-伊红染色（H&E）和Masson三色染色用于评估骨再生情况。

结果表明，与空白对照组相比，PHFS + NIR + SMF 组在 4 周和 8 周时新骨形成明显增加（图 10a）。同样，成骨相关蛋白的免疫荧光染色显示，与空白对照组相比，PHFS + NIR + SMF 组中 ALP、COL-1 和 OCN 的表达量较高（图 10b，d）。在马森染色实验中，PHFS + SMF、PHFS + NIR 和 PHFS + NIR + SMF 组比空白对照组和 PHFS 组显示出更多的胶原蛋白形成。此外，马森染色结果显示，近红外加热刺激产生了更多的微血管（图 10a）。接下来的血管生成相关蛋白免疫荧光染色证实了这一发现。在 PHFS + NIR 组和 PHFS + NIR + SMF 组中，血管内皮生长因子、CD31 和 HIF-

的蛋白表达明显增加（图 10c，e）。此外，8 周的动物切片显示，体内植入支架的降解率与新骨形成率一致。为了探究各组植入支架的毒性，对手术后 4 周的大鼠心脏、肝脏、脾脏、肺脏和肾脏进行了 H&E 染色观察。
The staining results indicated the biosafety of implanted scaffolds, magnetic field stimulation, and near-infrared heating (Fig. S10).
染色结果显示了植入支架、磁场刺激和近红外加热的生物安全性（图 S10）。

3. Conclusion 3.结论

In summary, a 4D printing shape memory scaffold containing

nanoparticles, PLGA and

was synthesized in this study. The scaffold possesses excellent shape memory and is capable of thermal and magnetic responses to near-infrared stimulation and static magnetic field stimulation, respectively. Using the dual-response mechanism, this scaffold enables minimally and precisely invasive implantation of personalized bone defects. Meanwhile, in vivo and in vitro experiments confirmed the synergistic effect of dual response on osteogenesis and angiogenesis. Transcriptomics analysis confirmed that this scaffold can upregulate the expression of the PI3K/AKT pathway through magnetic response, and the NIR thermal response further increased the phosphorylation of the AKT pathway by stimulating the high expression of HSP90, resulting in the high expression of downstream osteogenic and angiogenic related proteins. Our study provides a new strategy for minimally and precisely invasive treatment of bone defect repair: treatment of critical bone defects by means of 4D deformation and magnetic field navigation, magnetic field and near-infrared stimulation to promote osteogenesis and angiogenesis with minimally invasive implantation.
综上所述，本研究合成了一种含有

纳米粒子、PLGA 和

的 4D 打印形状记忆支架。该支架具有优异的形状记忆能力，并能分别对近红外刺激和静态磁场刺激产生热响应和磁响应。利用这种双重响应机制，该支架可实现个性化骨缺损的微创和精确植入。同时，体内和体外实验证实了双重响应对骨生成和血管生成的协同作用。转录组学分析证实，该支架可通过磁响应上调 PI3K/AKT 通路的表达，而近红外热响应通过刺激 HSP90 的高表达进一步提高了 AKT 通路的磷酸化，从而导致下游成骨和血管生成相关蛋白的高表达。我们的研究为骨缺损修复的微创和精确治疗提供了一种新策略：通过四维形变和磁场导航、磁场和近红外刺激治疗关键骨缺损，以微创植入促进成骨和血管生成。

4. Experimental section 4.实验部分

4.1. Materials 4.1.材料

PLGA (MW

kDa, Lactic: Glycolic

were purchased from Jinan Daigang Biomaterial Co., Ltd (Jinan, China). Hydroxyapatite nano-powders (MW

) were obtained from Macklin Co., Ltd (Shanghai, China).

nano-powders (

metals basis,

) were purchased from Innochem Co., Ltd (Beijing, China). Tetraethyl orthosilicate (TEOS) and ammonia solution (

) were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). Dichloromethane (DCM) was obtained from Tianjin Chemical Factory (Tianjin, China).
PLGA (MW

kDa, Lactic: Glycolic

购自济南大港生物材料有限公司（中国济南）。羟基磷灰石纳米粉体（MW

）购自麦克林有限公司（中国上海）。

纳米粉体（

金属基，

）购自英诺化学有限公司（中国北京）。正硅酸四乙酯（TEOS）和氨溶液 (

) 购自阿拉丁化学有限公司（中国上海）。二氯甲烷（DCM）购自天津化工厂（中国天津）。

4.2. Synthesis and Characterization of nanochains
4.2. 纳米链的合成与表征

The synthesis method of

nanochains were followed by Cijun Shuai (2022) [16]. In concrete terms, The synthesis principle is that the

nanoparticles are arranged in chains under the static magnetic field by the action of static magnetic field and the encapsulation of silica. The

nanoparticles were first dispersed in ethanolaqueous by ultrasonication for

, and then ammonia solution ( 28

) was added dropwise while stirring (

). TEOS was then added under stirring (

) and left for

. Finally, the formation of nanochains was induced in a static magnetic field. The synthetic

nanochains were observed by transmission electron microscope (TEM, JEM1400, Japan). The zeta potential of

nanoparticles were measured by a nano Zetasizer (Malvern ZEN3600, Britain). The crystal structures and compositions of

nanoparticles were detected by X-ray diffractometer (XRD, D8 Venture, Germany) and Fourier transform infra-red spectrometer (FTIR, VERTEX

, Germany). The magnetic properties of

nanoparticles were tested by vibrating sample magnetometer (VSM, LakeShore7404, America).

纳米链的合成方法由帅慈军（2022）[16]沿用。具体来说，其合成原理是通过静磁场的作用和二氧化硅的包裹，使

纳米粒子在静磁场下排列成链状。首先用超声波将

纳米粒子分散在乙醇水溶液中

，然后边搅拌边滴加氨水 ( 28

)(

)。然后在搅拌下加入 TEOS (

) 并静置

。最后，在静态磁场中诱导纳米链的形成。用透射电子显微镜（TEM，JEM1400，日本）观察合成的

纳米链。

纳米粒子的 zeta 电位由纳米 Zetasizer（Malvern ZEN3600，英国）测量。X 射线衍射仪（XRD，D8 Venture，德国）和傅立叶变换红外光谱仪（FTIR，VERTEX

，德国）检测了

纳米粒子的晶体结构和成分。用振动样品磁力计（VSM，LakeShore7404，美国）测试了

纳米粒子的磁性能。

4.3. Synthesis and characterization of 4D near-infrared responsive magnetic scaffolds
4.3.4D 近红外响应磁性支架的合成与表征

The weight ratio of PLGA to

was selected as

, and

was mixed according to

(group PH),

(group PHFSO.02),

(group PHFS0.04),

(group PHFS0.08), and

(group PHFS0.16) of the total PLGA/HA weight. First, PLGA,
选择 PLGA 与

的重量比为

，按照 PLGA/HA 总重量的

（PH 组）、

（PHFSO.02 组）、

（PHFS0.04 组）、

（PHFS0.08 组）和

（PHFS0.16 组）混合

。首先是 PLGA、

Fig. 9. In vivo bone regeneration assay of the scaffolds. a) Thermal images of the blank and PHFS + NIR groups with a power density of

for

. b) Thermal curves of the blank and PHFS + NIR groups. c) Schematic of the applied magnetic field (15mT) in PHFS + SMF and PHFS + NIR + SMF groups. d) Shape recovery of the PHFS scaffold under NIR stimulation (

). e) The quantitative analysis of bone regeneration in 4 weeks and 8 weeks post-operation, respectively. f) Micro-CT images of new bone regeneration in 4 weeks and 8 weeks post-operation, respectively.
图 9.a) 空白组和 PHFS + NIR 组的热图像，功率密度为

，

。 b) 空白组和 PHFS + NIR 组的热曲线。 c) PHFS + SMF 组和 PHFS + NIR + SMF 组的外加磁场（15mT）示意图。d) PHFS 支架在近红外刺激下的形状恢复 (

)。 e) 分别在手术后 4 周和 8 周的骨再生定量分析。 f) 分别在手术后 4 周和 8 周的新骨再生显微 CT 图像。

Fig. 10. In vivo assessments of bone regeneration in histological analysis. a) H&E and Masson staining of defected skulls of rats in 4 weeks and 8 weeks post-operation (HB: host bone, NB: new bone, dotted line: boundaries of the bone defects, yellow arrows: osteoblast, red arrows: microvascular). b) Immunofluorescence staining for osteogenesis-related proteins in rat cranial bones in 8 weeks post-surgery. c) Immunofluorescence staining for angiogenesis-related proteins in rat cranial bones in 8 weeks post-surgery. d) The corresponding semi-quantitative analysis of the Immunofluorescence staining for osteogenesis-related proteins in 8 weeks post-surgery. e) The corresponding semi-quantitative analysis of the Immunofluorescence staining for angiogenesis-related proteins in 8 weeks post-surgery.
图 10.a) 手术后 4 周和 8 周大鼠缺损头骨的 H&E 和 Masson 染色（HB：宿主骨，NB：新骨，虚线：骨缺损边界，黄色箭头：成骨细胞，红色箭头：微血管）。 b) 手术后 8 周大鼠颅骨骨生成相关蛋白的免疫荧光染色。c) 手术后 8 周大鼠颅骨血管生成相关蛋白的免疫荧光染色。

and

powders are dispersed in DCM solvent to form the bioprinting ink. Then, the designed STL file model was imported into a 3D extrusion printer (Regenovo 3D Bio-Architect, China) for printing. The scaffold is designed as a cylinder with a diameter of

, the wire spacing is set to

, the layer height is set to

(because of the melting and collapsing effect of setting the layer height for the nozzle diameter of

), the number of print layers is set to 4 layers, the diameter of printing nozzle is selected as

(22G), the printing speed is set to

, the air pressure is set to

. Final, the scaffolds were dried in a freeze dryer (FDU-2200, Japan) for

. The glass transition temperatures (

) of scaffolds were measured using a differential scanning calorimeter (DSC, Q20, America), and the scaffolds were heated from room temperature to

at a heating rate of

. Using a thermogravimetric analyzer (TAG, TGA550, America), the thermal behaviors of scaffolds were measured by heating the scaffolds from room temperature to

. The water contact angles of scaffolds were tested by an optical contact angle meter (OCA, Germany). The crystal structures and compositions of scaffolds were detected by XRD and FTIR. A universal testing machine (AGXPLUS10KN, Japan) was used to test the mechanical properties of scaffolds. The morphologies and elemental distributions of scaffolds were observed by scanning electron microscopy (SEM, FlexSEM1000,

和粉末分散在 DCM 溶剂中，形成生物打印墨水。然后，将设计好的 STL 文件模型导入三维挤出打印机（Regenovo 3D Bio-Architect，中国）进行打印。支架设计为直径为的圆柱体，线间距设置为，层高设置为（因为喷嘴直径为时设置层高会产生熔融和塌陷效应），打印层数设置为 4 层，打印喷嘴直径选择为（22G），打印速度设置为，气压设置为。最后，将支架在冷冻干燥机（FDU-2200，日本）中干燥。使用差示扫描量热仪（DSC，Q20，美国）测量支架的玻璃化转变温度（），并以的加热速率将支架从室温加热至。使用热重分析仪（TAG，TGA550，美国）测量了支架的热行为，将支架从室温加热到，加热速率为。用光学接触角仪（OCA，德国）测试了支架的水接触角。通过 XRD 和 FTIR 检测支架的晶体结构和成分。万能试验机（AGXPLUS10KN，日本）用于测试支架的机械性能。扫描电子显微镜（SEM，FlexSEM1000）观察了支架的形态和元素分布、

Japan) and energy-dispersive spectrometry (EDS, ZEISS EVO18, Germany). The degradation behaviors of scaffolds were measured by immersing the samples in phosphate buffered solution (PBS) at

for incubation and weighing every week for 8 weeks. The magnetic properties of scaffolds were tested by VSM. The scaffolds placed in a six-well plate were irradiated with near-infrared light and the heating rates of scaffolds were recorded with a photothermal camera. After heating the deformed scaffolds from room temperature to

, the degrees of shape recovery of scaffolds were observed and recorded.
日本）和能量色散光谱仪（EDS，ZEISS EVO18，德国）。将样品浸入磷酸盐缓冲溶液（PBS）中

进行培养，每周称重一次，连续 8 周，测量支架的降解行为。支架的磁性能通过 VSM 进行测试。用近红外光照射置于六孔板中的支架，并用光热照相机记录支架的加热速率。将变形的支架从室温加热到

后，观察并记录支架的形状恢复程度。

4.4. Cell culture 4.4.细胞培养

Young male Sprague-Dawley (SD) rats (5-7 days) were obtained from Yisi Experimental Animal Company (Changchun, China). Bone marrow was harvested from the femurs and tibias of SD rats (5-7 days old) and cultured in an incubator (

) for 2-4 generations. HUVECs were obtained from the State Key Laboratory of Oral Disease (changchun, China).
幼年雄性斯普拉格-道利（SD）大鼠（5-7 天）来自中国长春伊思实验动物公司。从 SD 大鼠（5-7 天大）的股骨和胫骨采集骨髓，在培养箱（

）中培养 2-4 代。HUVECs 取自口腔疾病国家重点实验室（中国长春）。

4.5. Cell proliferation assay
4.5.细胞增殖试验

rBMSCs of 3-4 generations were used for cell proliferation assay. The
使用 3-4 代的 rBMSCs 进行细胞增殖试验。细胞
proliferation of rBMSCs on scaffolds was evaluated by Cell Counting Kit8 (CCK-8, Beyotime Co., Ltd, China). After co-culturing the scaffolds with rBMSCs

cells/scaffold) for 1,3 , and 7 days, the scaffold samples were washed with PBS and then incubated with CCK-8 for

. Then,

of the incubated liquid was added to a 96 -well plate, and the absorbance of the solution was detected at

using a microplate reader.
用细胞计数试剂盒8（CCK-8，中国贝奥天时有限公司）评估支架上rBMSCs的增殖情况。支架与 rBMSCs

细胞/支架）共培养 1、3 和 7 天后，用 PBS 冲洗支架样品，然后用 CCK-8 孵育

。然后，将

培养液加入 96 孔板中，使用微孔板阅读器在

检测溶液的吸光度。

4.6. Cell viability assay
4.6.细胞活力检测

rBMSCs of 2-3 generations were used for cell viability assay. rBMSCs (

cells/scaffold) were implanted on the scaffolds for incubating. Cell viability on the scaffolds was assessed using a live/dead staining kit (Beyotime Co., Ltd, China). At the indicated times (1, 3, and 7 days), the medium was removed, cells were detached from the scaffolds, and washed three times with PBS. Subsequently, cells were stained with a live/dead staining kit for

. Finally, the stained cells were observed by a confocal laser scanning microscope (CLSM, Olympus FV3000, Japan).
将 2-3 代的 rBMSCs（

cells/scaffold）植入支架进行培养。使用活/死染色试剂盒（中国贝奥天美有限公司）评估支架上的细胞活力。在指定时间（1 天、3 天和 7 天）去除培养基，将细胞从支架上剥离，并用 PBS 冲洗三次。随后，用活体/死体染色试剂盒对细胞进行染色，

，

。最后，用激光共聚焦扫描显微镜（CLSM，日本奥林巴斯 FV3000）观察染色细胞。

4.7. Cell morphology 4.7.细胞形态

rBMSCs (

cells/scaffold) were implanted on the scaffolds for incubating. Phalloidin-Rhodamine (Beyotime, China) and DAPI (Solarbio, China) were used to evaluate the morphology of rBMSCs on the scaffolds for 7 days. After the scaffolds were fixed with paraformaldehyde, the cytoskeletal protein F-actin was stained with Phalloidin-Rhodamine (red) and the nuclei was stained with DAPI (blue). Three-dimensional images of the scaffolds were observed with a confocal laser scanning microscope (CLSM, Olympus FV3000, Japan).
将 rBMSCs（

cells/scaffold）植入支架进行培养。用 Phalloidin-Rhodamine （贝奥迪，中国）和 DAPI （索拉比奥，中国）评估支架上 7 天的 rBMSCs 形态。用多聚甲醛固定支架后，用Phalloidin-Rhodamine（红色）染色细胞骨架蛋白F-肌动蛋白，用DAPI（蓝色）染色细胞核。用激光共聚焦扫描显微镜（CLSM，日本奥林巴斯 FV3000）观察支架的三维图像。

4.8. In vitro osteogenic capacity assay
4.8.体外成骨能力试验

An osteogenic induction medium (

-MEM medium with

FBS, 1

penicillin-streptomycin,

-sodium glycerophosphate and 25

ascorbic acid) was used to promote osteogenic differentiation of rBMSCs on scaffolds. Cells and scaffolds were immersed in the osteogenic induction medium and the medium was changed every 3 days. The SMF group of scaffolds was applied to continuous magnetic stimulation (15mT), and the NIR group was applied to thermal stimulation

every two days, with each heating time of

. For the qualitative of alkaline phosphatase (ALP) assay, rBMSCs and scaffolds were cocultured in 12 -well plates (

cells/scaffold). After culturing the cells with the osteogenic induction medium for 7 and 14 days, the rBMSCs in each group were washed with PBS (3 times) and fixed with 4

paraformaldehyde for

, and then the rBMSCs in the well plates were stained with BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) according to the instructions. For the quantitative of alkaline phosphatase (ALP) assay, after the co-culture of rBMSCs and scaffolds for 7 and 14 days, cells were collected and evaluated with Alkaline Phosphatase Assay Kit (Beyotime, China) and BCA protein assay (Beyotime, China). In addition, the generation of calcium mineralization nodules during osteogenesis period was assessed by alizarin red (AR) staining. The rBMSCs and scaffolds (

cells/scaffold) were co-cultured in 6 -well plates with the osteogenic induction medium for 28 days to induce osteogenic differentiation. Then, the rBMSCs were fixed with

paraformaldehyde for

and stained with

alizarin red (Solarbio, China) solution (

) for

. Besides, osteogenic gene expression (COL-1, OCN, RUNX2, and BMP-2) evoked by scaffolds and stimuli was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR)

). The usage of RT-qRCT primers was shown in Table S1. The rBMSCs and scaffolds (

cells/scaffold) were co-cultured in 6-well plates with the osteogenic induction medium for 7 and 14 days. According to the instruction, TRIzol (Takara, Kusatsu, Japan) and Takara Reverse Transcriptase kit (Takara, Osaka, Japan) were used to extracted cellular RNA of rBMSCs and reverse-transcribed.
成骨诱导培养基（

-MEM培养基，含

FBS、1

青霉素-链霉素、

-甘油磷酸钠和25

抗坏血酸）用于促进支架上rBMSCs的成骨分化。将细胞和支架浸泡在成骨诱导培养基中，每 3 天更换一次培养基。SMF组支架接受连续磁刺激（15mT），NIR组每两天接受一次热刺激

，每次加热时间为

。为了进行碱性磷酸酶（ALP）定性检测，将 rBMSCs 和支架共培养在 12 孔板中（

cells/scaffold）。用成骨诱导培养基分别培养 7 天和 14 天后，用 PBS 冲洗各组 rBMSCs（3 次）并用 4

多聚甲醛固定

，然后用 BCIP/NBT 碱性磷酸酶显色试剂盒（百优天，中国）按说明对孔板中的 rBMSCs 进行染色。碱性磷酸酶（ALP）的定量检测是在 rBMSCs 与支架共培养 7 天和 14 天后，收集细胞并用碱性磷酸酶检测试剂盒（百优天公司）和 BCA 蛋白检测试剂盒（百优天公司）进行评估。此外，还采用茜素红（AR）染色法评估成骨过程中钙矿化结节的生成情况。将rBMSCs和支架（

cells/scaffold）置于6孔板中用成骨诱导培养基共培养28天，以诱导成骨分化。然后，用

多聚甲醛固定 rBMSCs（

），并用

茜素红（Solarbio，中国）溶液（

）染色（

）。此外，通过实时定量聚合酶链反应（RT-qPCR）

）评估了支架和刺激诱发的成骨基因（COL-1、OCN、RUNX2 和 BMP-2）表达。RT-qRCT 引物的用法见表 S1。将 rBMSCs 和支架（

cells/scaffold）在 6 孔板中用成骨诱导培养基共同培养 7 天和 14 天。根据说明书，使用 TRIzol（Takara，Kusatsu，Japan）和 Takara 逆转录酶试剂盒（Takara，Osaka，Japan）提取 rBMSCs 的细胞 RNA 并进行逆转录。
Then, the concentration of RNA was evaluated by Thermo NANODROP 2000c (Thermo Fisher Scientific, Fremont, CA). Final, the extracted RNA was reverse transcribed to cDNA by PrimeScript

RT-qPCR kit (Takara, Tokyo, Japan) and Applied Biosystems 7300 (ThermoScientific, Waltham, MA). Protein expression of ALP, COL-1, OCN and RUNX2 in cocultured rBMSCs was assessed by Immunofluorescence Staining. The rBMSCs and scaffolds (

cells/scaffold) were co-cultured in 12-well plates with the osteogenic induction medium for 7 days to induce osteogenic differentiation. Then, the rBMSCs were fixed with

paraformaldehyde for

and washed by PBS (3 times). Immunol staining blocking buffer (Beyotime, China), ALP Rabbit Monoclonal Antibody (ABclonal, China), COL-1 Rabbit Monoclonal Antibody (ABclonal, China), OCN Rabbit Monoclonal Antibody (ABclonal, China), RUNX2 Rabbit Monoclonal Antibody (ABclonal, China), and Alexa Fluor 488-labeled Goat Anti-rabbit IgG (Beyotime, China) were used for Immunofluorescence Staining following by the instruction. PhalloidinRhodamine (Beyotime, China) and DAPI (Solarbio, China) were used to stain the cytoskeleton and nuclei respectively. The images of Immunofluorescence Staining were captured by a stereo microscope (Olympus, Japan).
然后，用 Thermo NANODROP 2000c (Thermo Fisher Scientific, Fremont, CA) 评估 RNA 的浓度。最后，用 PrimeScript

RT-qPCR 试剂盒（Takara，Tokyo，Japan）和 Applied Biosystems 7300（ThermoScientific，Waltham，MA）将提取的 RNA 逆转录为 cDNA。免疫荧光染色法评估了共培养的 rBMSCs 中 ALP、COL-1、OCN 和 RUNX2 的蛋白表达。将 rBMSCs 和支架（

cells/scaffold）在 12 孔板中用成骨诱导培养基共培养 7 天，以诱导成骨分化。然后，用

多聚甲醛固定 rBMSCs，

，并用 PBS 冲洗（3 次）。免疫组化染色阻断缓冲液（贝奥天美，中国）、ALP 兔单克隆抗体（ABclonal，中国）、COL-1 兔单克隆抗体（ABclonal，中国）、OCN 兔单克隆抗体（ABclonal、RUNX2兔单克隆抗体（ABclonal，中国）和 Alexa Fluor 488 标记的山羊抗兔 IgG（Beyotime，中国）。PhalloidinRhodamine （百优天，中国）和 DAPI （Solarbio，中国）分别用于细胞骨架和细胞核染色。免疫荧光染色的图像由立体显微镜（日本奥林巴斯）拍摄。

4.9. Transcriptomics analysis of osteogenesis
4.9.成骨过程的转录组学分析

In order to investigate the possible molecular mechanisms associated with the role of PHFS + MIR + SMF group in osteogenic differentiation, gene expression profiling was used to compare the mRNA transcript levels among the groups (untreated control, PHFS + SMF group and PHFS + MIR + SMF groups). The rBMSCs and scaffolds

cells/ scaffold) were co-cultured in 6-well plates with the osteogenic induction medium for 7 days to induce osteogenic differentiation. Relevant pathways were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. For western blot (WB) experiment, the scaffolds and rBMSCs

) were co-cultured in 6 -well plates for 7 days. Then, cell lysis buffer for Western and IP (Beyotime, China) were used to extract proteins from the rBMSCs and the protein concentration in the extracts was calculated. Protein samples were added to a 4-20% gel (10180kD, Beyotime, China) and then the Tris-Glycine system was selected for electrophoresis. The final samples were colored using BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The results were analyzed semi-quantitatively with ImageJ software.
为了研究PHFS + MIR + SMF组在成骨分化过程中可能发挥作用的相关分子机制，采用基因表达谱比较了各组（未处理对照组、PHFS + SMF组和PHFS + MIR + SMF组）的mRNA转录水平。将 rBMSCs 和支架

cells/ scaffold）与成骨诱导培养基在 6 孔板中共培养 7 天，以诱导成骨分化。相关通路由京都基因组百科全书（KEGG）通路分析确定。在Western blot（WB）实验中，将支架和rBMSCs

)在6孔板中共同培养7天。然后用 Western 和 IP 用细胞裂解缓冲液（中国贝奥天美）提取 rBMSCs 中的蛋白质，并计算提取物中的蛋白质浓度。将蛋白质样品加入 4-20% 凝胶（10180kD，中国 Beyotime 公司），然后选择 Tris-Glycine 系统进行电泳。最后使用 BCIP/NBT 碱性磷酸酶显色试剂盒（中国 Beyotime）对样品进行显色。结果用 ImageJ 软件进行半定量分析。

4.10. In vitro angiogenic capacity assay
4.10.体外血管生成能力测定

Human umbilical vein endothelial cells (HUVECs) and scaffolds were co-cultured in 6-well plates for wound healing assay. Each well was implanted with

cells and cultured to

contact. The monolayer of cells was scratched with a

pipette tip and the detached cells were rinsed off with PBS. The residual cells were co-cultured with scaffolds in a high sugar medium (DMEM). The images were captured by an inverted phase microscope (DP74, OLYMPUS, Japan) and analyzed with ImageJ. The cell migration rate was calculated as follows

: Migration rate

is the range of the initial wound, and

is the remaining range of the wound at the measurement point

. For transwell assay, transwell cell with

pore size was used to culture the HUVECs (upper chamber) and scaffolds (lower chamber). After

, cells migrating into the lower chamber were fixed with

paraformaldehyde for

and then stained with crystal violet (Beyotime, China) for

. The images were captured by an inverted phase microscope (DP74, OLYMPUS, Japan) and the number of migrating HUVECs was calculated by ImageJ

. Matrigel basement membrane matrix was used to tube formation assay.

Matrigel/well was added to a 48 -well plate. Then, the 48-well plate was incubated in a cell incubator for 30 min until the matrix gel became solid. The cells

were resuspended in the extract solution of scaffolds and then seeded onto the Matrigel. After culturing HUVECs with Matrigel for 4-6 h, HUVECs in well plates were
将人脐静脉内皮细胞（HUVECs）和支架共同培养在 6 孔板中进行伤口愈合试验。每孔植入

细胞并培养至

接触。用

移液器吸头刮擦单层细胞，然后用 PBS 冲洗掉脱落的细胞。残余细胞在高糖培养基（DMEM）中与支架共同培养。用倒置相位显微镜（DP74，日本 OLYMPUS 公司）采集图像，并用 ImageJ 进行分析。细胞迁移率的计算方法如下

: 迁移率

。

是初始伤口的范围，

是测量点伤口的剩余范围

。对于 transwell 试验，使用孔径为

的 transwell 细胞培养 HUVEC（上腔）和支架（下腔）。

后，用

多聚甲醛固定移入下腔的细胞，

，然后用结晶紫（中国百代）染色，

。用倒置相位显微镜（DP74，日本 OLYMPUS 公司）拍摄图像，并用 ImageJ

计算迁移的 HUVEC 数量。用 Matrigel 基底膜基质进行试管形成试验。

在 48 孔板中加入 Matrigel/孔。然后，将 48 孔板放在细胞培养箱中培养 30 分钟，直到基质凝胶变成固体。将细胞

重悬于支架提取液中，然后播种到 Matrigel 上。用 Matrigel 培养 HUVEC 4-6 小时后，将孔板中的 HUVEC
stained with calcineurin AM and captured by a fluorescence microscope (Olympus, Japan). The average tube length of images was calculated by ImageJ software. Vascularization-related gene expression (VEGF, PDGF, HIF-

, and

-SMA) evoked by scaffolds and stimuli was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR)

). The usage of RT-qRCT primers was shown in Table S2. The HUVECs and scaffolds

cells/scaffold) were co-cultured in 6 -well plates for 3 days. According to the instruction, TRIzol (Takara, Kusatsu, Japan) and Takara Reverse Transcriptase kit (Takara, Osaka, Japan) were used to extracted cellular RNA of HUVECs and reverse-transcribed. Then, the concentration of RNA was evaluated by Thermo NANODROP 2000c (Thermo Fisher Scientific, Fremont, CA). Final, the extracted RNA was reverse transcribed to cDNA by PrimeScript

RT-qPCR kit (Takara, Tokyo, Japan) and Applied Biosystems 7300 (ThermoScientific, Waltham, MA). Protein expression of VEGF, HIF-

and CD31 in co-cultured HUVECs was assessed by Immunofluorescence Staining. The HUVECs and scaffolds were co-cultured in 12-well plates for 3 days. Then, the HUVECs were fixed with

paraformaldehyde for

and washed by PBS (3 times). Immunol staining blocking buffer (Beyotime, China), VEGF Rabbit Monoclonal Antibody (ABclonal, China), HIF-

Rabbit Monoclonal Antibody (ABclonal, China), CD31 Rabbit Monoclonal Antibody (ABclonal, China) and Alexa Fluor 488-labeled Goat Antirabbit IgG (Beyotime, China) were used for Immunofluorescence Staining following by the instruction. Phalloidin-Rhodamine (Beyotime, China) and DAPI (Solarbio, China) were used to stain the cytoskeleton and nuclei respectively. The images of Immunofluorescence Staining were captured by a stereo microscope (Olympus, Japan). For western blot (WB) experiment, the scaffolds and HUVECs

) were cocultured in 6 -well plates for 3 days. Then, cell lysis buffer for Western and IP (Beyotime, China) were used to extract proteins from the HUVECs and the protein concentration in the extracts was calculated. Protein samples were added to a 4-20 % gel (10-180kD, Beyotime, China) and then the Tris-Glycine system was selected for electrophoresis. The final samples were colored using BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The results were analyzed semiquantitatively with ImageJ software.
用钙调素 AM 染色，并用荧光显微镜（日本奥林巴斯）拍摄。图像的平均管长由 ImageJ 软件计算。通过实时定量聚合酶链反应（RT-qPCR）

）评估支架和刺激诱发的血管化相关基因表达（VEGF、PDGF、HIF-

和

-SMA）。RT-qRCT 引物的用法见表 S2。HUVECs 和支架

细胞/支架）在 6 孔板中共培养 3 天。根据说明书，使用 TRIzol（Takara，Kusatsu，Japan）和 Takara 逆转录酶试剂盒（Takara，Osaka，Japan）提取 HUVECs 的细胞 RNA 并进行逆转录。然后，用 Thermo NANODROP 2000c （Thermo Fisher Scientific, Fremont, CA）评估 RNA 的浓度。最后，用 PrimeScript

RT-qPCR 试剂盒（Takara，Tokyo，Japan）和 Applied Biosystems 7300（ThermoScientific，Waltham，MA）将提取的 RNA 逆转录为 cDNA。免疫荧光染色法评估了共培养的 HUVECs 中 VEGF、HIF-

和 CD31 的蛋白表达。HUVECs 和支架在 12 孔板中共培养 3 天。然后，用

多聚甲醛固定 HUVEC，

，并用 PBS 冲洗（3 次）。按照说明书使用免疫荧光染色阻断缓冲液（百优天，中国）、VEGF 兔单克隆抗体（百优天，中国）、HIF-

兔单克隆抗体（百优天，中国）、CD31 兔单克隆抗体（百优天，中国）和 Alexa Fluor 488 标记的山羊抗兔 IgG（百优天，中国）。细胞骨架和细胞核分别用类黄素（Phalloidin-Rhodamine）（百优天，中国）和 DAPI（Solarbio，中国）染色。免疫荧光染色图像由体视显微镜（日本奥林巴斯）拍摄。为了进行 Western blot（WB）实验，将支架和 HUVECs

) 共培养于 6 孔板中 3 天。然后用 Western 和 IP 用细胞裂解缓冲液（中国贝奥天美）从 HUVECs 中提取蛋白质，并计算提取物中的蛋白质浓度。将蛋白质样品加入 4-20 % 的凝胶（10-180kD，中国 Beyotime）中，然后选择 Tris-Glycine 系统进行电泳。最后使用 BCIP/NBT 碱性磷酸酶显色试剂盒（中国贝奥天美）对样品进行显色。结果用 ImageJ 软件进行半定量分析。

4.11. In vivo osteogenic capacity assay
4.11.体内成骨能力试验

SD rats (7-8 weeks old) were purchased from Yisi Experimental Animal Company (Changchun, China). All animal procedures were guided by the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the college of basic medicine of Jilin University (Permit Number:2023073). A schematic illustration of animal experiment is shown in Scheme 1b. All SD rats

) were divided into five groups randomly: Blank control, PHFS, PHFS + SMF, PHFS + NIR, PHFS + NIR + SMF. After anesthesia with

pentobarbital, two circular critical bone defects of

in diameter were created on the center of bilateral cranial parietal bone using a drill (

diameter). Then, each rat was injected with penicillin (

day) for 7 days. The thermal curves of the cranial defect sites in the blank control and NIR stimulation groups of the animals were tested using

laser at 2.5 W/

. In the group stimulated with NIR irradiation, rats received

NIR irradiation every 2 days, and the temperature was maintained at

for

. For the group with static magnetic field stimulation, magnetic stripes (

) were placed at intervals on the underside of each rat cage as show in Fig. 9c, and the magnetic field strength was determined to be

, which was measured by a digital Tesla meter (TD8620, Tunkia, China). At 4 and 8 weeks after surgery, anesthetized rats were executed and cranial specimens were collected. The samples were fixed in

paraformaldehyde for

. All samples were scanned with a micro-computed tomography scanner (mCT-50, SCANCO MEDICAL, Switzerland). After 3D reconstruction of the samples with Mimics Medical software, Scanco Medical analysis software was used to calculate and analyze bone volume fration (BV/ TV), bone surface (BS), trabecular number (Tb.N), trabecular separation
SD大鼠（7-8周龄）购自伊思实验动物公司（中国长春）。所有动物实验过程均遵循《吉林大学实验动物的饲养和使用准则》，并经吉林大学基础医学院批准（批准文号：2023073）。动物实验示意图见方案 1b。将所有 SD 大鼠

)随机分为五组：空白对照组、PHFS 组、PHFS + SMF 组、PHFS + NIR 组、PHFS + NIR + SMF 组。用

戊巴比妥麻醉后，用钻头（直径

）在双侧颅顶骨中心创建两个直径为

的圆形临界骨缺损。然后，每只大鼠连续 7 天注射青霉素（

day）。使用 2.5 W/

的

激光测试空白对照组和近红外刺激组动物颅骨缺损部位的热曲线。在近红外照射刺激组，大鼠每 2 天接受一次

近红外照射，温度保持在

，

。对于静态磁场刺激组，如图 9c 所示，在每个大鼠笼子的底部每隔一定距离放置磁条 (

) ，磁场强度由数字特斯拉计（TD8620，中国通家）测定为

。术后 4 周和 8 周，处死麻醉大鼠并采集颅骨标本。样本在

多聚甲醛中固定，

。用微型计算机断层扫描仪（mCT-50，瑞士 SCANCO MEDICAL 公司）扫描所有样本。使用 Mimics Medical 软件对样本进行三维重建后，Scanco Medical 分析软件用于计算和分析骨体积匀称度 (BV/TV)、骨表面 (BS)、骨小梁数 (Tb.N)、骨小梁分离度 (Tb.N)。
(Tb.Sp), and trabecular thickness (Tb.Th) at the defect site. All samples were decalcified with

EDTA for 4-5 weeks and sections were embedded. Then, the samples were stained with Masson trichrome staining and Hematoxylin-Eosin staining (H&E). Furthermore, immunofluorescence was performed on the tissue sections. Osteogenesisrelated antibodies (Anti- ALP, COL-1 and OCN) and angiogenesisrelated antibodies (Anti- VEGF, CD31 and HIF-1

) were applied to assess osteogenesis and angiogenesis at the defect sites in rats. All images were captured by an inverted microscope (Olympus, Japan). Besides, hearts, livers, spleens, lungs and kidneys of rats were collected at 4 weeks and then dehydrated and embedded. In vivo toxicity of the animals was evaluated by H&E staining.
(Tb.Sp）和缺损部位的小梁厚度（Tb.Th）。所有样本都用

EDTA 脱钙 4-5 周，然后将切片包埋。然后，用 Masson 三色染色法和 Hematoxylin-Eosin 染色法（H&E）对样本进行染色。此外，还对组织切片进行了免疫荧光。应用成骨相关抗体（Anti ALP、COL-1 和 OCN）和血管生成相关抗体（Anti VEGF、CD31 和 HIF-1

）评估大鼠缺损部位的成骨和血管生成情况。所有图像均由倒置显微镜（日本奥林巴斯）拍摄。此外，4 周后收集大鼠的心脏、肝脏、脾脏、肺脏和肾脏，脱水后包埋。用 H&E 染色法评估动物的体内毒性。

4.12. Statistical analysis
4.12.统计分析

All experiments were repeated at least three times and data results are presented as mean

standard deviation. GraphPad Prism 9 software was used to calculate and analyze the data by one-way analysis of variance (ANOVA). The statistically significant analysis results were indicated as

, ***p

, and

.
所有实验至少重复三次，数据结果以平均值

标准差表示。使用 GraphPad Prism 9 软件通过单因素方差分析（ANOVA）计算和分析数据。具有统计学意义的分析结果以

、***p

和

表示。

CRediT authorship contribution statement
CRediT 作者贡献声明

Yangyang Li: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jiaqian You: Software, Resources, Formal analysis, Data curation. Huixin Lv: Formal analysis, Data curation. Chong Wang: Writing - review & editing, Formal analysis, Data curation. Shaobo Zhai: Data curation. Sicong Ren: Data curation. Xiuyu Liu: Data curation. Yidi Zhang: Writing - review & editing, Writing original draft, Supervision, Resources, Funding acquisition, Formal analysis. Yanmin Zhou: Writing - review & editing, Supervision, Resources, Project administration, Funding acquisition.
李阳阳：写作--审阅和编辑、写作--原稿、可视化、验证、监督、软件、资源、项目管理、方法学、调查、形式分析、数据整理、概念化。游佳倩软件、资源、形式分析、数据整理。吕惠新形式分析、数据整理。王翀写作--审阅和编辑、形式分析、数据整理。翟少波数据整理任思聪：数据整理刘秀玉：数据整理张一迪写作--审阅和编辑、撰写原稿、督导、资源、资金获取、形式分析。周艳敏写作--审阅和编辑、指导、资源、项目管理、资金获取。

Declaration of competing interest
利益冲突声明

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有任何可能会影响本文所报告工作的已知经济利益或个人关系。

Data availability 数据可用性

Data will be made available on request.
数据将应要求提供。

Acknowledgements 致谢

This work was supported by the National Natural Science Foundation of China (Grant No. 82001092; Grant No. 82071152; Grant No. 82371006); Department of Education of Guangdong Province, China (2021ZDZX2014); and the Dongguan Science and Technology of Social Development Program, Guangdong, China (20211800904542). All animal proceedings were conducted under the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the College of Basic Medicine of Jilin University (Permit Number:2023073). Y.L. and J. Y. contributed equally to this work.
本研究得到了国家自然科学基金（批准号：82001092；批准号：82071152；批准号：82371006）、广东省教育厅（2021ZDZX2014）和广东省东莞市社会发展科技计划（20211800904542）的资助。所有动物实验均按照《吉林大学实验动物饲养与使用规范》进行，并经吉林大学基础医学院批准（许可证号：2023073）。Y.L.和J. Y.对本研究做出了同等贡献。

Appendix A. Supplementary data
附录 A.补充数据

Supplementary data to this article can be found online at https://doi. org/10.1016/j.cej.2024.151205.
本文的补充数据可在线查阅：https://doi. org/10.1016/j.cej.2024.151205。

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- Corresponding authors at: Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling, Hospital of Stomatology, Jilin University, Changchun 130021, Jilin, China.
  通讯作者吉林大学口腔医院吉林省牙齿发育与骨重塑重点实验室，吉林长春 130021。
E-mail addresses: zhangyidi@jlu.edu.cn (Y. Zhang), zhouym@jlu.edu.cn (Y. Zhou).
电子邮件地址：zhangyidi@jlu.edu.cn (Y. Zhang), zhouym@jlu.edu.cn (Y. Zhou)。

Y. Li. and J. You. contributed equally to this work.
Y.Li和J.You对本研究做出了同样的贡献。
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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

A R T I C L E I N F O

Keywords: 关键词：

Abstract 摘要

1. Introduction 1.导言

2. Results and discussion2.结果和讨论

2.1. Design of PLGA/HA/Fe scaffold2.1.PLGA/HA/Fe 支架的设计

2.2. Synthesis and Characterization of @SiO nanoparticles2.2. @SiO 纳米粒子的合成与表征

2.3. Fabrication and Characterization of 4D near-infrared responsive magnetic scaffolds2.3.四维近红外响应磁性支架的制作与表征

2.4. In vitro biocompatibility2.4.体外生物相容性

2.5. In vitro osteogenesis evoked by SMF and NIR2.5.SMF 和近红外诱发的体外骨生成

2.6. Transcriptomics analysis and western blotting (WB) assay of osteogenesis2.6.成骨的转录组学分析和西部印迹（WB）检测

2.7. In vitro angiogenesis evoked by SMF and NIR2.7.SMF 和近红外诱发的体外血管生成

2.8. In vivo bone regeneration2.8.体内骨再生

3. Conclusion 3.结论

4. Experimental section 4.实验部分

4.1. Materials 4.1.材料

4.2. Synthesis and Characterization of nanochains4.2. 纳米链的合成与表征

4.3. Synthesis and characterization of 4D near-infrared responsive magnetic scaffolds4.3.4D 近红外响应磁性支架的合成与表征

4.4. Cell culture 4.4.细胞培养

4.5. Cell proliferation assay4.5.细胞增殖试验

4.6. Cell viability assay4.6.细胞活力检测

4.7. Cell morphology 4.7.细胞形态

4.8. In vitro osteogenic capacity assay4.8.体外成骨能力试验

4.9. Transcriptomics analysis of osteogenesis4.9.成骨过程的转录组学分析

4.10. In vitro angiogenic capacity assay4.10.体外血管生成能力测定

4.11. In vivo osteogenic capacity assay4.11.体内成骨能力试验

4.12. Statistical analysis4.12.统计分析

CRediT authorship contribution statementCRediT 作者贡献声明

Declaration of competing interest利益冲突声明

Data availability 数据可用性

Acknowledgements 致谢

Appendix A. Supplementary data附录 A.补充数据

References 参考资料

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

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

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

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

2.4. In vitro biocompatibility
2.4.体外生物相容性

2.5. In vitro osteogenesis evoked by SMF and NIR
2.5.SMF 和近红外诱发的体外骨生成

2.6. Transcriptomics analysis and western blotting (WB) assay of osteogenesis
2.6.成骨的转录组学分析和西部印迹（WB）检测

2.7. In vitro angiogenesis evoked by SMF and NIR
2.7.SMF 和近红外诱发的体外血管生成

2.8. In vivo bone regeneration
2.8.体内骨再生

4.2. Synthesis and Characterization of nanochains
4.2. 纳米链的合成与表征

4.3. Synthesis and characterization of 4D near-infrared responsive magnetic scaffolds
4.3.4D 近红外响应磁性支架的合成与表征

4.5. Cell proliferation assay
4.5.细胞增殖试验

4.6. Cell viability assay
4.6.细胞活力检测

4.8. In vitro osteogenic capacity assay
4.8.体外成骨能力试验

4.9. Transcriptomics analysis of osteogenesis
4.9.成骨过程的转录组学分析

4.10. In vitro angiogenic capacity assay
4.10.体外血管生成能力测定

4.11. In vivo osteogenic capacity assay
4.11.体内成骨能力试验

4.12. Statistical analysis
4.12.统计分析

CRediT authorship contribution statement
CRediT 作者贡献声明

Declaration of competing interest
利益冲突声明

Appendix A. Supplementary data
附录 A.补充数据