在微创植骨领域，可注射骨水泥常用于修复微小且不规则的骨缺损。临床上使用的磷酸钙骨水泥（CPC），由于其密实结构及较差的生物降解性，限制了组织长入。本研究制备的基于含镁微球可注射骨水泥，其独有的3D多孔结构及可调的生物降解特性均有利于骨组织长入，同时其降解过程中释放出的镁离子能够发挥抗炎免疫成骨功能。
可注射骨水泥由于其优异的可塑性被广泛地用于填充和修复骨科创伤，尤其适用于形状不规则的骨缺损。然而，临床上最常用的可注射骨水泥，无论是生物惰性的聚甲基丙烯酸甲酯 (PMMA)骨水泥，还是生物活性的磷酸钙水泥(CPC)，都难以原位形成相互连通的多孔结构。多孔结构可以为细胞附着、长入以及随后的成骨分化和血管重塑提供空间。若缺乏多孔结构，血管长入和新骨形成将完全依赖于骨水泥自身的降解。目前商业化CPC具有较低且不可控的生物降解性，这使得CPC成为密实的块体，新的骨组织仅沿着CPC边缘生长，难以长入其内部。为了解决这些问题，特别是降解速率慢的问题，研究者们已经尝试将CPC与磷酸镁水泥(MPC)混合制备复合骨水泥。
MPC因其快速固化、高强度和快速生物降解的特点而受到关注。更重要的是，作为人体的必需元素，镁元素参与了许多重要的生理过程，包括维持激素水平、免疫响应及促进骨形成等。另一方面，巨噬细胞在种植体诱导的免疫反应中也发挥重要作用。通常，巨噬细胞可以被激活为促炎表型的M1巨噬细胞，或抗炎表型的 M2巨噬细胞，发挥免疫调节作用，使组织由炎症过程向重建过程转变。调节巨噬细胞极化，以恢复M1/M2表型的平衡或主要向M2表型极化有利于取得良好的骨植入修复效果。先前的研究表明，含镁的骨植入物，如 β-TCP包覆的镁金属及MgSiO3 包覆的羟基磷灰石，可以引起 M2巨噬细胞极化及随后的骨生成。这些发现不仅表明了镁在种植体-骨相互作用中的重要性，而且预示了含镁骨水泥也可能引起M2巨噬细胞主导的免疫响应并增强骨再生。因此，将 MPC添加到CPC中，预期不仅会引起较短的固化时间和良好的生物降解性，而且还会由于 Mg离子的免疫调节发挥更好的促骨生成作用。
由于CPC和MPC的起始原料是粉末，固化后形成的复合骨水泥仍无法形成连通的多孔结构。因此，为了获得3D多孔结构，必须引入额外的致孔过程，例如 NaCl 颗粒浸出致孔等。尽管这些方法具有一定的效果，但是固化后的骨水泥仍然是块体结构。迄今为止，尚未见有含镁的可注射骨水泥可以原位固化成多孔结构且兼具良好生物降解性的报道。
近年来，3D打印和其他增材制造技术在组织工程领域显示出独特的优势。在我们之前的研究中，使用选择性激光烧结技术可以实现PCL和磷酸钙复合微球接触界面的融合联结，从而得到任意形状的多孔支架。受此启发，我们提出将含镁可注射骨水泥的前体成形为微球，以期制备可原位固化的3D多孔支架。微球形态可以赋予含镁可注射骨水泥良好的流变性能，从而提高可注射性。固化反应可以被限制在微球的接触界面上，从而减慢反应速度并减少放热。微球之间的间隙可形成连通孔，为细胞迁移和组织浸润留出空间，进一步通过生物降解可为更多的组织长入提供便利。更重要的是，降解释放出的镁离子能够发挥免疫调节作用，促进巨噬细胞向M2型极化，从而触发血管和新骨生成。（Fig.1）
Fig. 1. Schematic illustration for the principles of bone defects filling and repair by MMSC. The MMSs are not only of the desired physicochemical properties to meet the requirement of injectable bone cement for clinical application, but also can be cured into a 3D interconnected porous scaffold conducive to cells and tissue ingrowth in situ. Moreover, the controllable biodegradation of MMSC continuously provides increasing space while the release of magnesium ions induces a tissue repair favourable immunoregulation via the M2 phenotype polarization of macrophages for the consecutive vascularization and new bone formation.
在此，本研究发展了一种基于含镁微球 (MMS) 的可注射骨水泥。MMSs的主要成分是 MgO，与 MPC 相同，这保证了MMSs 骨水泥 (MMSC) 的快速固化。其次要成分是 MgSiO4 和 Ca7Mg2P6O24。MgSiO4可以维持骨水泥原位固化后的微球结构，而生成的Ca7Mg2P6O24 消耗了部分氧化镁，可以减少MMSC 固化过程中的放热。研究了MMSC的物理化学特性，包括可注射性、放热特性、离子释放和生物降解性，并与 CPC和MPC 进行了比较。我们还通过炎症基因表达和巨噬细胞极化研究了MMSC中Mg离子的免疫调节功能。随后使用RAW264.7细胞和大鼠骨髓间充质干细胞 (rbMSCs) 的共培养系统研究了MMSC浸提液对成骨分化的影响。最后通过皮下植入模型，在体内验证MMSC可以诱导组织长入、发挥成骨免疫调节作用，并促进骨生成。
实验结果表明，通过液滴冷凝法及烧结处理得到了具有多孔结构的含镁微球，且其主要成分为氧化镁。此外，主相为氧化镁的含镁微球可以在磷酸二氢氨溶液中正常固化，其固化过程是微球间生成片状磷酸镁氨结晶。（Fig.2和Fig.3）
Fig. 2. Preparation and characterization of MMSs. (a) Schematic diagram of the MMSs fabrication process. (b) SEM showed that micropores distributed on the surface and in the interior of the MMSs. (c) BET results indicated that the pore diameters of MMSs mainly ranged from 2~60 nm. (d) The elemental mapping results displayed two distribution areas: Ca-P and Mg-Si-O. (e) XRD patterns showed that the main phase of MMSs was MgO, and the secondary phases were Ca7Mg2P6O24 and Mg2SiO4 compared with JCPDS#45-0946, JCPDS#20-0348, and JCPDS#34-0189, which corresponded to MgO, Ca7Mg2P6O24, and Mg2SiO4, respectively.
Fig. 3. Setting process of MMSC. (a) Digital photography and SEM images exhibited interconnected porous scaffolds with different 3D shapes cured by MMSC. (b) XRD results indicated that MgO within MMSs had reacted completely, and a new phase, NH4MgPO4.6H2O, formed after setting for 12 h compared with JCPDS#15-0762. (c) SEM image at a high magnification showed lamellar crystals formed between microspheres after setting for 20 min. (d) Element line scan results indicated that the phase composite of the lamellar crystals was NH4MgPO4.6H2O; 1, 2, and 3 pointed to the phases of MgO/Mg2SiO4, Ca7Mg2P6O24 and NH4MgPO4.6H2O, respectively. (e) Schematic diagram of the MMSC setting process.
研究了含镁微球骨水泥（MMSC）其物理化学性能，包括流变性、固化过程中的放热、固化时间、初期力学强度等。结果表明，其物理化学性能和使用性能均能满足临床上对可注射骨水泥的要求。另外，相比CPC和MPC，MMSC具有可控的生物降解和钙镁离子释放特性。（Fig.4）
Fig. 4. Physiochemical properties of MMSC compared with those of the MPC and CPC. (a) The rheological curve showed that the MMSC pastes had a constant and low viscosity, which suggested good rheological properties and practicable injectability. (b) The heat release of MMSC was well-controlled in comparison with MPC suggesting that the microspheres effectively reduced the exothermic reaction (c) The setting time was approximately 10 min in the MMSC, which well-met the requirement of clinical application. (d) Mechanical strength results showed that the compressive strength of the MMSC was stable after setting for 1 h. (mean ± SD; n = 3; *significant difference compared with the MPC group, *p < 0.05, **p < 0.01; # significant difference between groups, #p < 0.05). (e) MMSC had a moderate degradation rate with a weight loss of approximately 8 wt% after 21 days (mean ± SD, n=5). (f, g) Calcium and magnesium ions can be released from the MMSC simultaneously at a moderate rate (mean ± SD, n=5).
为了探究MMSC是否可以促进巨噬细胞向M2型极化及对间充质干细胞（rbMSCs）成骨分化的影响，首先研究了MMSC浸提液的细胞毒性，结果显示当浸提液浓度为3.125mg/mL时可以促进巨噬细胞增殖，且对rbMSCs无细胞毒性。随后研究了RAW264.7巨噬细胞对此MMSC浸提液的免疫响应，结果表明RAW264.7巨噬细胞中的抗炎症基因CD206和IL-10上调，并分泌较多的抗炎蛋白IL-10，这说明MMSC浸提液可以促进巨噬细胞向M2型极化。进一步的RT-PCR实验结果显示经MMSC浸提液培养后的RAW264.7巨噬细胞和rbMSCs共培养后，还促进了rbMSCs中成骨相关基因COL1的上调。（Fig.5）
Fig. 5. In vitro cytocompatibility, immunoregulation and osteogenic induction. (a, b) CCK-8 assay showed all cement extracts with concentration ≤ 3.125 mg/mL were of no cytotoxicity to RAW264.7 cells. B: DMEM; 128, 64, 32, and 16 represent 1/128, 1/64, 1/32 and 1/16 of the original extract concentration (200 mg/mL), respectively. (c) Cement extracts with a concentration of 3.125 mg/mL had no cytotoxicity on rbMSCs. (d, e) RT-PCR results showed that the relative expression levels of CD206 and IL-10 significantly upregulated in RAW264.7 cells treated with MMSC extract at a concentration of 3.125 mg/mL, whereas the relative expression levels of IL-6 were significantly higher than IL-10 in the MPC and CPC groups. (f) ELISA tests revealed that the concentration of IL-10 in the MMSC group was significantly higher than that in the B and MPC group in the coculture system of RAW264.7 cells and rbMSCs. (g) RT-PCR results demonstrated that the relative expression level of COL1 in the MMSC group was upregulated and significantly higher than that in the B group in the coculture system (mean ± SD; n = 3; *significant difference compared with the B group, *p < 0.05, **p < 0.01, ***p < 0.001; # significant difference between groups, #p < 0.05).
随后，研究了新材料的体内炎症响应。结果表明：植入初期，MMSC引起中度炎症反应，同时出现明显的组织长入和血管生成。这归因于其本身的多孔结构以及降解过程中产生的孔隙。进一步的免疫组化染色结果显示，植入14天后，MMSC引起的炎症逐渐减弱，并使巨噬细胞极化为较多的M2型巨噬细胞，这与体外实验结果一致。（Fig.6和Fig.7）
Fig. 6. Short-term immune response in vivo. Masson staining of MPC, CPC and MMSC subcutaneously implanted for 3 days (a, b, c) and 7 days (d, e, f). White dash lines portray the material boundaries. Green pentagrams indicate the cells infiltrated into the materials via the biodegradation of MPC and MMSC. White arrows point out the newly formed blood vessels in the gap of the MMSs. Scale bars are 50 μm. (g-l) SEM with elemental mapping images indicated the tissue ingrowth after 7 days of material implantation through the distribution of carbon elements (marked in red). White dotted lines describe the material boundaries. Scale bars are 500 μm. (m, n, o) HE staining of MPC, CPC and MMSC after 14 days of implantation. Aggregation of massive immune cells was observed surrounding the incompletely degraded MPC, while the CPC with no obvious degradation was encapsulated by compact fibrous tissues. By contrast, continuous ingrowth of cells and blood vessels took the space provided by the degradation of the microspheres in MMSC.
Fig. 7. Immunostaining of CD68, F4/80, CD86 and CD163 in the (a) MPC, (b) CPC and (c) MMSC after 14 days of material implantation. Red arrows pointed out the CD163-positive stained cells. CD68, F4/80, CD86 and CD163 are the cell markers of the immune cells, macrophages, M1 macrophages and M2 macrophages, respectively. The positive staining of these markers was found surrounding the MPC and CPC while inside the MMSC, demonstrating the porous structure of MMSC was beneficial to the inward migration of immune cells and macrophages. More importantly, CD163 was much more positive than CD86 in MMSC, which suggested a predominant M2 phenotype polarization of macrophages conducive to anti-inflammation and tissue repair induced by the MMSC.
最后，通过异位成骨模型，研究了MMSC的骨生成能力。结果显示，植入12周后，MMSC逐渐降解，并生成大量的新生骨，且无明显的纤维组织包裹。这说明相比CPC和MPC，MMSC更有利于骨生成。这可能是由于MMSC固化后的3D多孔结构、可控的生物降解性及镁离子引起的抗炎免疫调节共同促进了骨生成（Fig.8）
Fig. 8. Subcutaneous ectopic osteogenesis after 12 weeks of cements implantation. (a-f) No obvious new bone formation was found within the MPC and CPC, while they were encapsulated by dense fibrous membranes. (g-k) There was not only no obvious fibrous membrane encapsulating the MMSC, but also massive new bone formation was observed within the MMSC. Osteoblasts were also noticed at the boundaries of the incompletely degraded MMSs. M: material; NB: new bone; OB: osteoblast; Dotted circle: microspheres with new bone
综上述，本研究发展了一种含镁微球可注射骨水泥新体系MMSC及其制备策略，并用于抗炎免疫调节促进骨再生。体内外实验表明，MMSC不仅能满足临床上对于可注射骨水泥的基本要求，还可以在固化后原位形成3D多孔结构，更有利于组织长入。同时，其降解过程中释放出的镁离子能够发挥抗炎免疫调节作用，促进骨生成。这预示着MMSC在骨组织修复和再生领域的重要应用前景。
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博士，华中科技大学先进生物材料与组织工程研究中心。研究方向为用于骨修复生物材料。
博士，华中科技大学先进生物材料与组织工程研究中心。研究方向为用于治疗骨肿瘤的生物材料。
博士、华中科技大学先进生物材料与组织工程研究中心讲师，硕士生导师，入选第六届中国科协青年人才托举工程计划。主要从事骨、软骨修复生物材料及组织工程支架研究，在Science Advances, Chemical Reviews, Biomaterials, Bioactive Materials, Advanced Healthcare Materials等高水平国际期刊发表第一作者/通讯作者论文；先后主持承担国家自然科学基金面上项目、NSFC青年项目及中国博士后科学基金特别资助项目，以第二负责人完成国家自然科学基金国际合作重点项目，作为主要研究骨干参与多项国家重点研发计划项目课题的研究；作为副主编组织编写了“双**”交叉学科研究生高水平课程教材《高等生物材料学实验》一部；指导本科生参加全国大学生生物实验技能竞赛并获得全国一等奖。
博士、澳大利亚昆士兰科技大学教授。肖殷教授主要兴趣在于利用生物材料与组织工程等方法技术促进骨缺损修复及骨相关疾病的治疗；多年来在Nature Communications, Biomaterials, Advanced Functional Materials等发表260余篇SCI论文，被引用超4000余次；撰写专著两部，同时参与编写书籍章节10余部；获得37项研究基金资助；受邀担任8个国际学术期刊的编委，应邀组织承办国际组织工程和再生医学学会（TERMIS）等多个国际**学术会议。
博士、教授/博士生导师、国际生物材料科学与工程院Fellow (FBSE)、TERMIS亚太当选主席、中国生物材料学会副理事长、国家重点研发计划首席科学家、中国科协首席科学传播专家；华中科技大学医疗器械监管科学研究院院长、华中科技大学先进生物材料与组织工程交叉学科中心主任、国际再生医学材料联合实验室主任。兼任CFDA/NMPA医疗器械审评技术专家咨询委员会委员、CFDA医疗器械分类技术委员会委员。Advanced Healthcare Materials、Tissue Engineering、Biomedical Materials等期刊编委或特邀主编。
张胜民教授在先进再生医学材料、生物3D打印专用生物材料、生物能量活性材料、多分子模板仿生矿化、功能元素摻杂钙磷生物材料等领域做出了系统性、开创性贡献。是国际再生医学材料系列大会、全国再生医学材料系列大会两大学术交流平台的创始人和联合主席、国际组织工程系列大会共同主席。主持承担国家重点研发计划、国家自然科学基金重点项目、国家自然科学基金国际合作重点项目、国家863计划项目、国家科技支撑计划项目、国家重大研究计划项目、科技部政府间国际科技合作计划项目等30余项， 获得中国和美国发明专利授权30余项，部分重大发明获得转化，取得中国CFDA/NMPA和美国FDA医疗器械产品注册证5项，产生显著社会经济效益。在Science Advances, Chemical Reviews, ACS Nano, Biomaterials, Biotechnology Advances, Bioactive Materials等期刊发表学术论文100余篇。2016年，因其“在再生医学材料研发、 临床转化和促进生物材料科学公共进步领域所作出的公认的杰出贡献”，而被授予国际生物材料科学与工程学会联合会会士（IUSBSE Fellow， FBSE）终身荣誉称号。
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上述研究工作得到了国家重点研发计划项目（2018YFC1105701），国家自然科学基金（81801850, 81901897, 31870960）及中国博士后科学基金（2018M642851）资助。
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Shenglong Tan1, Yifan Wang1, Yingying Du*, Yin Xiao* and Shengmin Zhang*.
Injectable bone cement with magnesium-containing microspheres enhances osteogenesis via anti-inflammatory immunoregulation.
Bioactive Materials, 2021, 6: 3411-3423.
