Small RNA-Modulated Exosome Mimetics For Craniofacial Regeneration

用于颅面再生的小 RNA 调节外泌体模拟物

• 批准号: 10394941
• 负责人: Jiabing Fan
• 金额: $ 2.29万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2022-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394941
• 关键词: Affect; Allografting; Animal Model; Apatites; Autologous; BMP2 gene; Bone Regeneration; Caffeic Acids; Cell Transplantation; Cells; Cephalic; Characteristics; Chronic; Clinical; Communication; Craniofacial Abnormalities; Data; Defect; Degenerative Disorder; Deposition; Development; Disease; Drug Carriers; Drug Kinetics; Excision; Exhibits; Face; Feedback; Foundations; Future; Gelatin; Generations; Goals; Growth Factor; Head; Histologic; Homeostasis; Homing; Human; Immobilization; Impairment; Implant; Label; Mandible; Measurement; Mediating; Medical; Mesenchymal Stem Cells; Metabolism; MicroRNAs; Minerals; Modeling; Morbidity - disease rate; Mus; Natural regeneration; Operative Surgical Procedures; Osteoblasts; Play; Process; Production; Property; Quality of life; Rattus; Regulation; Research; Rodent; Role; Signal Transduction; Site; Small Interfering RNA; Small RNA; Stains; Stimulus; Structure; Surgical Flaps; Therapeutic; Transfection; Transport Process; Trauma; Treatment Efficacy; Up-Regulation; Vesicle; Work; X-Ray Computed Tomography; antagonist; base; bone; bone healing; clinical practice; comparison group; cost; craniofacial; craniofacial bone; craniofacial repair; dosage; exosome; extracellular vesicles; fluorescence imaging; healing; immunogenicity; improved; microCT; migration; mimetics; novel strategies; novel therapeutics; osteogenic; paracrine; prevent; public health relevance; reconstruction; regenerative; repaired; response; restoration; scaffold; skeletal; skeletal regeneration; skeletal stem cell; tissue regeneration; tumor

Abstract Large craniofacial defects remain an extraordinary challenge to clinical surgical reconstruction. Conventional approaches of auto/allografting for clinical craniofacial reconstruction are significantly compromised by availability and donor-site morbidity. Mesenchymal stem cells (MSCs) with multipotency are increasingly employed as a cell-based approach for skeletal regeneration. Nonetheless, accumulative evidences suggest that contribution of MSCs to regenerated tissues is limited while stimulation of local healing processes through paracrine secretion exerts more important roles. MSC-derived extracellular vesicles (EVs) have shown regenerative potency in varying animal models and displayed therapeutic advantages like intrinsic homing effect, stability, low immunogenicity and effective signaling stimulation. However, the widespread use of exosome-mediated treatment still requires the significant improvement of production yield and regenerative ability. Developing a scalable approach like generation of exosome mimetics (EMs) with substantial yields has been investigated in previous studies. Additionally, exosome-mediated cargo of exogenous therapeutic factors like siRNAs has been conducted to improve its regenerative capability. The exogenous transport of these factors is also growing concerns of high cost, poor pharmacokinetics and inefficiency. Herein, the augmentation of intrinsic inductive/therapeutic molecules within exosomes exhibits a promising therapeutic strategy. Skeletal cells secrete important growth factors like BMP2, which incite osteoblastic commitment of skeletal progenitor cells and subsequent mineral deposition. In response to BMP stimulus, MSCs or osteoblasts elevate BMP antagonist like noggin, suggesting a negative feedback to prevent overexposure of BMP signaling. Introduction of exogenous noggin was revealed to impair cranial formation while inhibition of endogenous noggin promoted cranial regeneration by activating endogenous BMP signaling. These observations highlight the potency of noggin suppression on up-regulation of endogenous BMP activity and subsequent osseous deposition. In our preliminary studies, EMs were generated from MSCs via a distinctive extrusion approach, demonstrating high yields of exosomes with apparent osteogenic induction. Moreover, EMs obtained from noggin-suppression MSCs (EM-NG) revealed the elevated noggin siRNA and osteogenic potency. Together, we hypothesize that EM-NG could enhance osteoblastic commitment of endogenous skeletal stem cells (SSCs) and craniofacial regeneration. Two specific aims are proposed to investigate this hypothesis: 1) To investigate the effect of EM-NG on endogenous cranial SSCs and bone healing; 2) To examine the implant of EM-NG-laden scaffold for restoration of segmental mandibular defects. The completion of this proposal will offer significant foundation to further develop effective cell-free approaches for clinical craniofacial defect repair. The additional roles of EMs on craniofacial restoration will be further explored in large animal model in future. These future studies will be proposed in the subsequent R01 application.

抽象的 大颅面缺损仍然是临床手术重建的巨大挑战。传统的 用于临床颅面重建的自体/同种异体移植方法受到以下因素的严重影响： 可用性和供体部位发病率。具有多能性的间充质干细胞（MSC）越来越多 用作基于细胞的骨骼再生方法。尽管如此，累积的证据表明 间充质干细胞对再生组织的贡献是有限的，而通过刺激局部愈合过程 旁分泌发挥着更重要的作用。 MSC 衍生的细胞外囊泡 (EV) 已显示 在不同的动物模型中具有再生能力，并显示出内在归巢等治疗优势 效果好、稳定性好、免疫原性低、信号刺激有效。然而，广泛使用 外泌体介导的治疗仍需要显着提高产量和再生能力 能力。开发一种可扩展的方法，例如产生具有可观产量的外泌体模拟物（EM） 在之前的研究中进行过调查。此外，外泌体介导的外源治疗因子货物 类似的 siRNA 已被用来提高其再生能力。这些物质的外源运输 人们越来越担心成本高、药代动力学差和效率低等因素。在此，将 外泌体内内在诱导/治疗分子的增强表现出一种有前途的治疗方法 战略。骨骼细胞分泌重要的生长因子，如 BMP2，它会刺激成骨细胞的定向 骨骼祖细胞和随后的矿物质沉积。响应 BMP 刺激，MSC 或 成骨细胞会升高 BMP 拮抗剂（如 noggin），这表明存在负反馈以防止过度暴露 BMP 信号传导。研究表明，引入外源性头蛋白会损害颅骨形成，同时抑制 内源性头蛋白通过激活内源性 BMP 信号传导促进颅骨再生。这些 观察结果强调了头蛋白抑制对内源性 BMP 活性上调的效力， 随后的骨沉积。在我们的初步研究中，EMs 是通过独特的 MSCs 产生的 挤出方法，证明外泌体产量高，具有明显的成骨诱导作用。而且， 从头蛋白抑制 MSC (EM-NG) 获得的 EM 显示头蛋白 siRNA 和成骨作用升高 效力。我们共同假设 EM-NG 可以增强内源性成骨细胞的承诺 骨骼干细胞（SSC）和颅面再生。提出了两个具体目标来调查这一问题 假设：1）研究EM-NG对内源性颅骨SSC和骨愈合的影响； 2) 至 检查装有 EM-NG 的支架的植入物，以修复节段性下颌骨缺损。这 该提案的完成将为进一步开发有效的无细胞方法提供重要基础 临床颅面部缺损修复。将进一步探讨 EM 对颅面修复的额外作用 未来在大型动物模型中。这些未来的研究将在后续的 R01 申请中提出。