微生物合成的聚羟基脂肪酸酯（PHA）材料由于具有优良的生物相容性和生 物可降解性，因此在组织修复与再生领域得到广泛的关注。然而，大部分 PHA 材 料均存在生物活性较弱、结构保守、不易修饰等问题，极大限制了其在组织修复 与再生领域的应用。本论文通过物理共混、化学改性等方法实现了 PHA 材料的功 能化，并分别在体外细胞水平和体内动物水平对其进行了功能性验证。 首先，使用静电纺丝技术将 50μg mL-1抗生素环丙沙星（CIP）和（0.5、1.0、 2.0mg mL-1）促血管生成剂（DMOG）分别共混入聚（3-羟基丁酸-co-4-羟基丁酸） 材料（P34HB）中，形成了抗菌和促血管生成的功能化 P34HB 多孔纤维膜。在全 皮肤缺损模型中，功能化 P34HB 多孔纤维膜在 14 天的伤口愈合率可达到 90%， 远远高于 P34HB 的 47%和 P34HB/CIP 组的 53%。通过活体血管光声显微成像 （OR-PAM）实验发现，在 14 天时间内，在 P34HB 多孔纤维膜的新生组织中仅观 察到较密集的新生血管，而功能化多孔纤维膜中的血管生成则已到达重塑阶段， 即血管再次融合形成成熟的血管网络。 此外，本论文还开发了一种简单、方便的功能化修饰技术。首先利用熔融打 印技术并结合低温成型方法，实现孔径可调的（从 300μm 到 800μm）3D 支架打印 制备。通过聚多巴胺（PDA）的作用将 31.5%的具有骨生成促进功能的 BMP2 修 饰到 3D 多孔 P34HB 支架上。体外成骨诱导分化实验表明，功能化支架促进了人 源脂肪间充质干细胞（ADSC）的成骨分化相关基因的表达，提高了胞外钙结节的 沉积能力。大鼠临界性颅骨缺损实验结果表明，BMP2 修饰的 3D 多孔 P34HB 支 架相比于 P34HB 支架，其骨缺损部分新生骨的密度（BMD）、骨小梁数量（Tb.N） 等指标均是实验组中最高的。 工程改造微生物合成带有较高反应活性碳碳双键的 PHA 共聚物，是实现 PHA 多元化功能修饰的另一种有效策略。本论文一方面通过调整 PHA 凝胶的交联程度， 对其压缩强度进行精准调控；另一方面通过探究材料的结构与性能关系，为小口 径血管制备提供了科学基础。 综上所述，本论文通过对 PHA 材料的功能化修饰，提高了皮肤再生过程中的 血管生成能力，促进了骨缺损部位骨组织再生和功能重建。而 PHA 作为支架修复 材料的简便的功能化修饰方法建立，进一步提升其力学和生物学性能，拓宽了 PHA 材料在生物医学领域的应用潜力和商业价值。
The microbial synthesized Polyhydroxyalkanoates (PHAs) have been studied for a variety of applications in tissue engineering owning to their biocompatible and biodegradeable nature. However, many disadvantages of PHAs, such as the poor biological activity, highly conservative structures and modification difficulties, have significantly limited the applications of inducing tissue repair and regeneration. Here, we thus report the functionalized PHAs fabrication by employing physical blending and chemical modification, which demonstrated expected functionality in vitro (cell level) and in vivo tests (animal level). Firstly, electrospinning was conducted to generate a functionalized P34HB porous fiber membrane with strong antibacterial properties by blending poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) with two active small molecules, 50μg mL-1 of antibiotic ciprofloxacin (CIP) and 0.5, 1.0, 2.0mg mL-1 of pro-angiogenesis agent (DMOG), respectively, to induce the formation of blood vessels form vascular endothelial cells (HUVECs). Compared to the P34HB porous fibrous membrane containing 1.0mg mL-1 DMOG, which displayed greater capability in promoting angiogenesis in vitro, the functionalized P34HB porous fibrous membrane also demonstrated excellent ability to promote wound healing in the mouse skin defect model. Notably, the wound healing rate of 14-days of functionalized P34HB group reached up to 90%, which is almost 2-fold higher than the control groups of P34HB and P34HB/CIP, reaching only 47% and 53%, respectively. Photoacoustic experiments in vivo showed that, within 14 days, only a large number of capillaries were observed in the blood vessels of newly formed tissue in the P34HB porous fibrous membrane, however, the angiogenesis in the functionalized porous fibrous membrane reached fully remodeling stage, forming mature vascular networks. Besides, a simple and convenient functional modification method was developed in this study using the melt printing technology combined with low-temperature molding method, namely low-temperature 3D printing technology, to generate P34HB scaffolds with the customized pore size from 200 to 800 μm. Subsequently, the 3D printed P34HB scaffolds were modified with BMP2, an active protein that can promotes bone formation, through the connection of polydopamine (PDA) with loading efficiency of 31.5%. Interestingly, the osteogenic differentiation experiments in vitro showed that the BMP2 functionalized scaffold can significantly promote the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSC). Results of the rat skull defect experiment of BMP2 functionalized P34HB scaffold also displayed the best performances in bone mineral density (BMD), trabecular number (Tb.N) and other factors in contrast to the control group, P34HB scaffold with null modification. Moreover, the synthesis of vinyl PHAs with higher reactivity carbon-carbon double bonds by engineered Psedomonas entomophlia is an effective strategy to achieve founctionalized PHAs via chemical modification. Here, two cases were studied to demostrate the diverse application of vinyl PHAs: 1) The compressive strength of PHA gel can be tuned precisely by adjusting the cross-linking degree; 2) The characterization of relationship between material structures and properties provides a theoretical basis for the downstream preparation of small-caliber blood vessels. In summary, several approaches have been developed to generated functionalized PHAs for different modification purposes, which successfully promotes angiogenesis in skin regeneration, induces the repair and functional reconstruction of bone tissue in situ, respectively, with promising performances. The low-temperature 3D printing method of novelty also makes PHA as a powerful scaffold biomaterial with tailor-made mechanical and biological properties for tissue regeneration, which can significantly expand the commercial applications and values of PHA in biomedical uses in the future.
