钛表面稳定结合、刚性可调载基因纳米囊泡聚电解质膜构建及促进骨结合机制研究

Osseointegration is one of important markers indicating the success of implants. The osseointegration can be improved by coating polyelectrolyte multilayer films (PEMs) on implant surfaces. But most of films are bonded with implants by weak linkages including ionic bonds and or hydrogen bonds, resulting in the weak film-substrate bonding strength, and the film stiffness is much lower than the bone tissue. The suitable film-substrate bonding strength is a basic prerequisite to guarantee that films will not be broken during implanting and will function after implanting. The film stiffness can modulate the growth of cells and bacteria, and cells usually favor substrates with stiffness equivalent to themselves. Our preliminary data show that: PEMs with distinctive three-dimensional nanostructure can be constructed on titanium surfaces by using pBMP2-gene-loaded lipopolysaccharide-amine nanopolymersomes as a cationic polyelectrolyte, and hyaluronic acid as an anionic polyelectrolyte. The film can slowly release gene and in situ transfect bone marrow mesenchymal stem cells (MSCs), significantly facilitate the adhesion, proliferation and differentiation of MSCs, and finally enhance osseointegration, indicating their great potential for clinic applications. Based on these, this project aims to enhance the bonding strength between the PEM and titanium substrate, to improve the film stiffness, and to further optimize the gene release by using catechol groups to modify the above mentioned polyelectrolytes. The effects of the film-substrate bonding strength, film stiffness and in situ gene transfection on film stability, in vitro bacterium/cell growth and in vivo osseointegration will be studied. The relevant mechanism will be explored. The project aims to provide the theory and experiment basis for the design of surface modification on dental implants from mechanics and biology.

骨结合是种植体植入成功的重要标志。种植体表面聚电解质多层膜改性可提高骨结合能力。但目前膜-种植体间多通过离子键、氢键等连接，膜基结合力较弱，且膜刚性远低于骨组织。而适宜的膜基结合强度是保证膜在植入时不受干扰或破坏，在植入后稳定发挥功能的基本前提；膜刚性则是调控细胞、细菌生长的重要因素，细胞更青睐和自身刚度相当的基底环境。我们前期研究发现：以透明质酸、载BMP2基因的可降解脂多糖胺纳米囊泡分别为阴、阳离子聚电解质，在钛表面能构建具有独特三维纳米结构的多层膜，其能缓释基因并原位转染骨髓间充质干细胞,显著提高细胞的粘附、增殖、分化，促进骨结合，预示临床应用的巨大潜能。本课题拟在此基础上，采用儿茶酚改性聚电解质以提高膜基结合力和膜刚性、进一步优化基因释放速度，研究前述因素对膜稳定性、体外细菌/细胞生长及体内骨结合影响，进而阐明其中机制，从力学和生物学角度为牙种植体表面改性设计提供理论与实验依据。

本项目围绕牙种植体表面膜改性对骨结合的影响及机理进行研究。采用儿茶酚化透明质酸（cHA）、载BMP-2基因（或siNoggin）的脂多糖胺纳米囊泡（NPs）分别为阴、阳离子聚电解质，儿茶酚化NPs（cNPs）为引发层，通过层层自组装方法在钛表面构建了儿茶酚化聚电解质多层膜（cPEM）。利用cNPs中的儿茶酚基团将膜固定在钛表面，使其拥有稳定的膜基结合；再利用cHA中的儿茶酚基团和 NPs表面大量氨基反应交联膜，提高其刚性；通过改变儿茶酚浓度优化膜理化性能，获得具适宜膜基结合力、刚性、降解速度，并能通过原位基因转染在种植体周围长效制造骨生长因子的膜，从而为骨生长提供适宜微环境。结果表明，在所研究范围内，当cNPs表面氨基、cHA上羧基的儿茶酚取代度分别为40%、10%时，膜力学性能最佳：膜基结合强度达38.62 mN，能耐受植入时机械损伤，基本不产生剥脱；弹性模量约为14.03 GPa，与天然骨组织（4～27GPa）相当，能促进成骨相关细胞粘附、增殖、生长，并一定程度抑制变形链球菌粘附。膜能长效释放负载的基因并转染细胞制造靶蛋白；囊泡载siNoggin较载BMP-2基因更能促进细胞成骨分化。大鼠体内植入实验表明，膜改性较未改性钛种植体更能促进骨结合，表现为种植体表面骨量和生物拔出力增加等。综上，我们基本验证了本课题的猜想并达到预期目标，即cPEM膜能通过儿茶酚进一步提高膜粘附和内聚作用，实现在种植体表面的稳定结合并获得适宜刚性，再结合基因治疗作用，从力学、分子生物学等多方面调控骨生长过程，促进了种植体骨结合，具临床应用潜能，预期能为种植体表面改性的设计制造提供理论实践依据。迄今前述研究已发表13篇论文（5篇SCI），另有1篇SCI在投；获授权1项发明专利；获广东医学科学技术奖三等奖；参加7次学术会议（境外3次），培养3名研究生。