Facile Osteoimmunomodulatory Nanoparticles Augment 3D-Printed Scaffold-Mediated Bone Regeneration

Regenerating critical-sized long bone defects poses substantial challenges due to limitations of autografts and processed allografts. Biomaterial scaffolds offer versatile alternatives, yet their effectiveness is often constrained by their limited innate osteoinductivity. While growth factors and cells can enhance osteoinduction, the inclusion of biologics in biomaterial scaffolds creates regulatory challenges for clinical translation. To address this, here we describe three-dimensional (3D) printed polycaprolactone (PCL) scaffolds for temporally controlled delivery of osteoimmunomodulatory amorphous calcium phosphate-chitosan nanoparticles (ACPC-NP). In vitro, the ACPC-NP exhibit concentration dependent effects on osteoblasts, monocytes, and osteoclasts. At increasing concentrations up to 500 μg/ml, these nanoparticles stimulate osteogenesis, modulate M2/M1 macrophage polarization, and inhibit osteoclast maturation and activity. Leveraging these concentration-dependent effects in vivo through temporally controlled release of ACPC-NP from 3D-printed PCL scaffolds, we observe the complete regeneration and the restoration of biomechanical strength of critically sized radial defects in rats. Such healing is absent in defects implanted with bare PCL scaffolds or those loaded with calcium-phosphate microparticles. The tunable osteoimmunomodulation by the NP underscore the translational potential of this technology to yield structurally sound and functionally robust bone regeneration outcomes. Three types of scaffolds were fabricated using 3D printing techniques, including PCL scaffolds containing amorphous calcium phosphate chitosan nanoparticles (ACPC-NP), calcium phosphate microparticles (CaP), and bare PCL. We then set out to test their effects in vivo using a rat radius critical-sized bone defect model. To gain mechanistic insights on the osteogenic capacity of the ACPC-NP scaffold compared to CaP and PCL scaffolds, bulk RNA sequencing was performed on mRNA from cells released from the bone tissues extending 1mm proximal and distal to the scaffolds at weeks 2 and 6 post-implantation.

临界尺寸长骨缺损的再生治疗面临诸多严峻挑战，这源于自体骨移植与处理后同种异体骨移植的固有局限。生物材料支架虽提供了多样化的替代方案，但其疗效常受限于先天骨诱导能力不足。尽管生长因子与细胞可增强骨诱导能力，但在生物材料支架中引入生物制剂会给临床转化带来监管层面的难题。

为解决这一问题，本研究报道了一种三维（3D）打印聚己内酯（polycaprolactone, PCL）支架，用于骨免疫调控型无定形磷酸钙-壳聚糖纳米颗粒（amorphous calcium phosphate-chitosan nanoparticles, ACPC-NP）的定时控释。体外实验表明，ACPC-NP对成骨细胞、单核细胞及破骨细胞的作用呈浓度依赖性：当浓度升至500 μg/ml时，该纳米颗粒可促进成骨、调控M2/M1巨噬细胞极化，并抑制破骨细胞的成熟与活性。

借助3D打印PCL支架实现ACPC-NP的定时控释，利用上述浓度依赖性效应开展体内实验，我们观察到大鼠临界尺寸桡骨缺损实现了完全再生，并恢复了缺损部位的生物力学强度。仅植入裸PCL支架或负载磷酸钙微粒（calcium phosphate microparticles, CaP）的缺损组则未出现此类骨愈合现象。该纳米颗粒所具备的可调控骨免疫调节能力，凸显了本技术的转化潜力，有望实现结构完整、功能强健的骨再生效果。

本研究通过3D打印技术制备了三类支架：负载ACPC-NP的PCL支架、负载CaP微粒的PCL支架，以及裸PCL支架。随后，我们采用大鼠桡骨临界尺寸骨缺损模型，在体内验证这三类支架的作用效果。为深入解析ACPC-NP支架相较于CaP与PCL支架的成骨能力机制，我们在植入后第2周和第6周，对支架近端及远端1mm范围内骨组织释放的细胞总mRNA进行了批量RNA测序。