Li Zhe, Qu Tiejun, Ding Chen, Ma Chi, Sun Hongchen, Li Shirong, Liu Xiaohua
Department of Plastic and Aesthetic Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, People's Republic of China; Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX 75246, USA.
Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX 75246, USA; Department of Operative Dentistry & Endodontics, State Key Laboratory of Military Stomatology, School of Stomatology, Fourth Military Medical University, Xi'an 710032, People's Republic of China.
Acta Biomater. 2015 Feb;13:88-100. doi: 10.1016/j.actbio.2014.11.002. Epub 2014 Nov 8.
Injectable biomaterials are attractive for soft tissue regeneration because they are handled in a minimally invasive manner and can easily adapt to complex defects. However, inadequate vascularization of the injectable constructs has long been a barrier, leading to necrosis or volume reduction after implantation. In this work, we developed a three-step process to synthesize injectable gelatin-derived hydrogels that are capable of controlling growth factor delivery to induce angiogenesis. In our approach, tyramine was first introduced into gelatin chains to provide enzymatic crosslinking points for gel formation after injection. Next, heparin, a polysaccharide with binding domains to many growth factors, was covalently linked to the tyramine-modified gelatin. Finally, vascular endothelial growth factor (VEGF) was incorporated into the gelatin derivative by binding with the heparin in the gelatin derivative, and an injectable gel with controlled VEGF release was formed by an enzymatic catalytic reaction with hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The gelation time, mechanical properties and degradation of the gel was readily tailored by the gelatin concentration and the ratio of H2O2/HRP. Binding VEGF to heparin stabilizes this growth factor, protects it from denaturation and proteolytic degradation and subsequently prolongs the sustained release. An in vitro release study and bioactivity assay indicated that the VEGF was released in a sustained manner with high bioactivity for over 3 weeks. Furthermore, a chicken chorioallantoic membrane (CAM) assay and animal experiments were performed to evaluate in vivo bioactivity of the VEGF released from the hydrogels. After 5 days of incubation on CAM, the number of blood vessels surrounding the heparin-modified hydrogels was increased by 2.4-fold compared with that of the control group. Deeper and denser cell infiltration and angiogenesis in the heparin-modified gelatin/VEGF gels were observed compared to the controls after being subcutaneously injected in the dorsal side of the mice for 2 weeks. Interestingly, even without the incorporation of VEGF, the heparin-modified gelatin derivative still had the capability to induce angiogenesis to a certain degree. Our results suggest that the gelatin derivative/VEGF is an excellent injectable delivery system for induced angiogenesis of soft tissue regeneration.
可注射生物材料因其以微创方式操作且能轻松适应复杂缺损而对软组织再生具有吸引力。然而，可注射构建体的血管化不足长期以来一直是一个障碍，导致植入后坏死或体积减小。在这项工作中，我们开发了一种三步法来合成可注射的明胶衍生水凝胶，其能够控制生长因子的递送以诱导血管生成。在我们的方法中，首先将酪胺引入明胶链中，以提供注射后凝胶形成的酶交联点。接下来，肝素，一种与许多生长因子具有结合域的多糖，与酪胺修饰的明胶共价连接。最后，血管内皮生长因子（VEGF）通过与明胶衍生物中的肝素结合而掺入明胶衍生物中，并且通过与过氧化氢（H2O2）和辣根过氧化物酶（HRP）的酶催化反应形成具有可控VEGF释放的可注射凝胶。凝胶的凝胶化时间、机械性能和降解可通过明胶浓度和H2O2/HRP的比例轻松调整。将VEGF与肝素结合可稳定这种生长因子，保护其免于变性和蛋白水解降解，并随后延长持续释放。体外释放研究和生物活性测定表明，VEGF以持续方式释放，具有高生物活性超过3周。此外，进行了鸡胚绒毛尿囊膜（CAM）试验和动物实验以评估从水凝胶释放的VEGF的体内生物活性。在CAM上孵育5天后，肝素修饰水凝胶周围的血管数量与对照组相比增加了2.4倍。在小鼠背部皮下注射2周后，与对照组相比，在肝素修饰的明胶/VEGF凝胶中观察到更深和更密集的细胞浸润和血管生成。有趣的是，即使不掺入VEGF，肝素修饰的明胶衍生物仍具有一定程度诱导血管生成的能力。我们的结果表明，明胶衍生物/VEGF是用于软组织再生诱导血管生成的优异可注射递送系统。
