生物活性硅酸钙复合壳聚糖水凝胶改善炎症微环境促进牙周组织再生的研究

The inflammatory microenvironment is the main bottleneck for periodontal tissue regeneration. The development and application of biomaterials with antibacterial, anti-inflammatory and tissue regeneration capacities is of great importance for periodontal regeneration. Among various biomaterials, injectable chitosan hydrogel has received major attention in periodontal tissue regeneration due to its antimicrobial activity, anti-inflammatory effects, and good formability to fill irregular periodontal defects. However, traditional methods for chitosan hydrogel preparation using either chemical or physical crosslinking of chitosan chains are potentially cytotoxic. In our previous studies, an injectable enzymatically cross-linked chitosan hydrogel (eCH) was developed and evaluated for periodontal regeneration. This hydrogel is highly biocompatible, biodegradable, and improves periodontal regeneration in terms of functional ligament length. However, the effect and mechanism of eCH in the inflammatory response are unknown, and the mechanical and osteostimulative properties of eCH still needs to be improved. Silicon has been considered as an agent for promoting the formation of new bone. Recently, as a strength-enhancing additive, calcium silicate nanowires (nCS) have attracted particular attention, since it enhances osteogenic and angiogenic differentiation. Thus, bioactive nCS will be incorporated into eCH to improve the mechanical and biological properties of eCH, and a novel injectable hybrid hydrogel nCS/eCH will be fabricated. The effect of nCS/eCH on periodontal ligament cells in terms of osteogenic and angiogenic differentiation will be evaluated in an inflammatory microenvironment. The antimicrobial and anti-inflammatory properties with the focus on the related mechanisms will be investigated. Furthermore, an inflammatory periodontal defect model will be applied for transplantation of nCS/eCH, and the in vivo periodontal tissue regeneration will be evaluated.

炎症微环境是影响牙周再生的关键瓶颈。研制兼具抗炎抗菌作用及促组织再生功能的生物材料对实现炎症微环境下的牙周再生具有重要意义。壳聚糖水凝胶具有抗炎抗菌作用且应用方便，但其传统制备方法有潜在细胞毒性。我们前期研制了新型的可注射壳聚糖酶促物理交联水凝胶（eCH），具有良好的生物相容性并能促进牙周韧带的再生，但其抗炎效应及机制仍然未知，成骨活性及力学性能有待改善。硅元素是促进新骨形成的一个媒介，硅酸钙纳米线（nCS）具有良好的成骨成血管活性及优良的力学增强性能。本项目拟将前期研制的eCH复合生物活性力学增强体nCS，制备可注射复合材料nCS/eCH并对其性能进行评价，揭示复合材料炎症微环境下对牙周膜细胞的成骨/成血管/抗炎效应并阐明其抗炎机制，明确复合材料对常见牙周致病菌的抗菌作用，并应用于动物炎性牙周缺损的修复，实现炎症微环境下牙周再生修复材料的制备及应用研究，具有重要的科学意义和临床应用价值。

炎症微环境是影响牙周再生的关键瓶颈。此外，牙周膜（PDL）细胞体外扩增过程中极易发生干性衰退，导致牙周再生能力降低，是影响其应用的关键瓶颈。研制兼具抗炎抗菌作用及促组织再生功能的生物材料并提升PDL细胞质量对实现炎症微环境下的牙周再生具有重要意义。壳聚糖水凝胶具有抗炎抗菌作用且应用方便，但其传统制备方法有潜在细胞毒性。我们前期研制了新型的可注射壳聚糖酶促物理交联水凝胶（eCH），具有良好的生物相容性并能促进牙周韧带再生。本研究旨在进一步改善eCH的成骨活性并提升牙周组织再生种子细胞质量，主要内容如下：（1）通过尿素与尿素酶的作用成功制备了一种纳米羟基磷灰石/壳聚糖酶促物理交联水凝胶（nHA/eCH），并对其凝胶时间、形貌结构及体外成骨诱导能力进行评价。这种新型的水凝胶可原位成胶，具有良好的生物相容性。含有nHA的eCH能够从模拟体液中吸收更多的钙，并且nHA/eCH能够促进PDL细胞钙盐沉积量，具有良好的生物活性，为牙周组织再生提供了一种新型的生物材料。（2）提供了一种更加有效的PDL细胞体外扩增方法。制备了一种更接近生理环境弹性模量值的聚二甲基硅氧烷（PDMS）组织培养基底，与传统的硬度更高的组织培养聚苯乙烯（TCPS）相比，在PDMS上培养的PDL细胞比TCPS培养的细胞具有更小的尺寸和更长的形态，更小的铺展面积、更少的焦点附着斑和更快的迁移速度。与TCPS相比，更接近生理硬度的PDMS基底培养在不影响细胞自我更新能力的情况下促进了PDL细胞的快速体外扩增。（3）发展了一种三维PDL细胞球体制备方法。证实了PDL细胞可以在壳聚糖膜上形成三维多细胞球体。与传统单层培养的细胞相比，三维PDL细胞球体具有更高的干性相关基因表达、克隆形成能力和成骨分化能力，有望作为更优质的种子细胞用于牙周组织再生。