Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo.
Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo.
复制标题
通过化学接枝修饰技术从壳聚糖支架中零级控制释放BMP2衍生肽P24，以促进体外成骨和增强体内骨修复
作者：
Chen Y;Liu X;Liu R;Gong Y;Wang M;Huang Q;Feng Q;Yu B
组织工程骨支架与细胞粘附、骨传导或骨诱导生物分子的结合是改善其显著影响细胞行为（如粘附、增殖和分化）的特性的关键策略，这有利于关键尺寸骨缺损的修复。然而，传统的表面改性技术，如物理吸附、涂层、等离子体处理等，由于其控制释放性能不理想或操作步骤过于复杂，使得生物活性分子的固定化受到很大的限制。本研究采用化学接枝改性技术，对壳聚糖/羟基磷灰石（CS/HA）仿生复合支架进行功能化修饰，以控制BMP 2衍生肽（P24）的释放：首先将P24与巯基化壳聚糖-4-硫代丁基脒（CS-TBA）偶联，然后将CS-P24与HA复合，制备CS-P24/HA支架。评价CS-P24/HA支架对骨再生的影响，沿着体外和体内相关的生物学机制。体外释放实验表明，活性成分P24的控释和缓释时间可达90 d，且CS-5%P24/HA和CS-10%P24/HA的释放曲线呈线性，符合零级动力学模型（R2=0.9929; R2=0.9757）;支架上的P24显著促进细胞粘附、增殖、骨分化和矿化，具有协同效应。骨髓基质细胞（BMSCs）显示纺锤形的表面形态，表明CS-P24/HA支架支持细胞粘附，并具有根据P24浓度水平而变化的高增殖率。此外，与体外在CS/HA支架上生长的细胞相比，OCN、Runx 2和胶原I的mRNA水平在CS-P24/HA支架上显著上调（p < 0.05）。与CS/HA支架相比，BMSCs在CS-P24/HA支架上表现出更高的ALP表达和钙沉积水平（p < 0.05）。体内实验结果显示，CS-10%P24/HA支架在大鼠背部肌袋内异位成骨的能力明显高于CS/HA支架。最后，CS-P24/HA支架在大鼠颅骨缺损的修复中表现出了上级的性能。这种新型的CS-P24/HA支架被认为是一种强有力的潜在候选人，用于修复人类骨组织工程中的骨缺损。
Combination of tissue-engineered bone scaffolds with cell-adhesive, osteoconductive, or osteoinductive biomolecules is a critical strategy to improve their properties that significantly influence cellular behaviors, such as adhesion, proliferation, and differentiation, which is beneficial for critical-sized bone defects repairing. However, the traditional surface modification techniques, such as physical adsorption, coating, and plasma treatment, et al, have great limitations for immobilization of bioactive molecules due to undesirable controlled delivery performance or overly complex multistep procedures. In this study, we functionalized the chitosan/hydroxyapatite (CS/HA) biomimetic composite scaffold for controlled delivery of BMP2-derived peptide (P24) by the chemical grafting modification technique: firstly, P24 was conjugated with a thiolated chitosan, chitosan-4-thiobutylamidine (CS-TBA); secondly, the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds. The effect of CS-P24/HA scaffolds on bone regeneration was evaluated, along with the underlying biological mechanisms responsible in vitro and in vivo. In vitro, the controlled and sustained release of bioactive P24 could last up to 90 days, furthermore, the release profiles of CS-5%P24/HA and CS-10%P24/HA were linear and could be fitted according to zero-order kinetic model (R2=0.9929; R2=0.9757); P24 on the scaffold significantly promoted cell adhesion, proliferation, osteodifferentiation, and mineralization with synergistic effects. Bone marrow stromal cells (BMSCs) revealed spindle-shaped surface morphology, indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate that varied according to the P24 concentration levels. Furthermore, mRNA levels for OCN, Runx2, and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro (p < 0.05). Similarly, the BMSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds (p < 0.05). In vivo, osteoinductive studies revealed a significantly higher ectopic osteogenesis level of CS-10%P24/HA scaffolds in rat dorsal muscle pockets compared with that of CS/HA scaffolds. Finally, CS-P24/HA scaffolds showed superior performance in the reconstruction of rat calvarial bone defects. This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in human bone tissue engineering.