Wang Xiaohong, Ackermann Maximilian, Wang Shunfeng, Tolba Emad, Neufurth Meik, Feng Qingling, Schröder Heinz C, Müller Werner E G
ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, D-55128 Mainz, Germany.
Biomed Mater. 2016 May 5;11(3):035005. doi: 10.1088/1748-6041/11/3/035005.
In this study the effect of amorphous calcium carbonate (ACC) microparticles and amorphous calcium polyphosphate (polyP) microparticles (termed aCa-polyP-MP) on bone mineral forming cells/tissue was investigated in vitro and in vivo. The ACC particles (termed ACC-P10-MP) were prepared in the presence of Na-polyP. Only the combinations of polyP and ACC microparticles enhanced the proliferation rate of human mesenchymal stem cells (MSCs). Gene expression studies revealed that ACC causes an upregulation of the expression of the cell membrane-associated carbonic anhydrase IX (CA IX; formation of ACC), while the transcript level of the alkaline phosphatase (ALP; liberation of orthophosphate from polyP) changes only relatively little. In contrast, aCa-polyP-MP primarily induces ALP expression. If both components are applied together a strong stimulation of expression of both marker genes is observed. In order to investigate whether ACC also enhances bone regeneration induced by polyP in vivo, the particles were encapsulated into PLGA (poly(d,l-lactide-co-glycolide)) microspheres (diameter ~800 μm) and implanted into rat critical-size calvarial defects. The studies revealed that animals that received aCa-polyP-MP microspheres showed an increased rate of regeneration compared to β-tri-calcium phosphate (β-TCP) controls. This effect is even accelerated if microspheres with both aCa-polyP-MP and ACC-P10-MP (1 : 1 weight ratio) are applied, resulting in an almost complete restoration of the defect area after 12 weeks. qRT-PCR analyses of tissue sections through the regeneration zone with microspheres containing both aCa-polyP-MP and ACC-P10-MP revealed a significantly higher upregulation of expression of the marker genes compared to each of the components alone. The Young's moduli for microspheres containing aCa-polyP-MP (1.74 MPa) and aCa-polyP-MP/ACC-P10-MP (2.38 MPa) were markedly higher compared to β-TCP-controls (0.63 mPa). Our results show that the combined application of ACC and Ca-polyP (both in the amorphous state) opens new strategies for the development of regenerative implants for the reconstruction of bone defects.
在本研究中，对无定形碳酸钙（ACC）微粒和无定形聚磷酸钙（聚P）微粒（称为aCa - polyP - MP）对骨矿物形成细胞/组织的影响进行了体外和体内研究。ACC颗粒（称为ACC - P10 - MP）是在Na - 聚P存在的情况下制备的。只有聚P和ACC微粒的组合提高了人间充质干细胞（MSCs）的增殖率。基因表达研究表明，ACC导致细胞膜相关碳酸酐酶IX（CA IX；ACC的形成）表达上调，而碱性磷酸酶（ALP；从聚P中释放正磷酸盐）的转录水平变化相对较小。相比之下，aCa - polyP - MP主要诱导ALP表达。如果将两种成分一起应用，则会观察到两种标记基因的表达受到强烈刺激。为了研究ACC在体内是否也能增强聚P诱导的骨再生，将颗粒封装到聚（d，l - 丙交酯 - 共 - 乙交酯）（PLGA）微球（直径约800μm）中，并植入大鼠临界尺寸的颅骨缺损处。研究表明，与β - 磷酸三钙（β - TCP）对照组相比，接受aCa - polyP - MP微球的动物显示出更高的再生率。如果应用含有aCa - polyP - MP和ACC - P10 - MP（重量比1∶1）的微球，这种效果会进一步加速，12周后缺损区域几乎完全恢复。对含有aCa - polyP - MP和ACC - P10 - MP的微球通过再生区的组织切片进行qRT - PCR分析发现，与单独的每种成分相比，标记基因的表达上调明显更高。含有aCa - polyP - MP（1.74MPa）和aCa - polyP - MP/ACC - P10 - MP（2.38MPa）的微球的杨氏模量明显高于β - TCP对照组（0.63mPa）。我们的结果表明，ACC和Ca - 聚P（均处于无定形状态）的联合应用为开发用于修复骨缺损的再生植入物开辟了新策略。
