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: 10196364
• 负责人: Jiabing Fan
• 金额: $ 15.6万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196364
• 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; 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 大型颅面缺陷仍然是对临床手术重建的巨大挑战。传统的 临床颅面重建的自动/同种异体方法被严重损害 可用性和供体现场发病率。具有多元的间充质干细胞（MSC）越来越多 用作基于细胞的骨骼再生方法。尽管如此，累积证据表明 MSC对再生组织的贡献受到限制，而刺激局部愈合过程 旁分泌的分泌发挥了更重要的作用。 MSC衍生的细胞外囊泡（EV）已显示 在不同的动物模型中的再生效力，并显示出诸如内在归住的治疗优势 效果，稳定性，低免疫原性和有效的信号刺激。但是，广泛使用 外泌体介导的治疗仍然需要显着提高生产率和再生 能力。开发一种可扩展的方法，例如具有实质产量的外泌体模拟物（EMS）具有 在先前的研究中进行了研究。另外，外源性治疗因素的外泌体介导的货物 像siRNA一样，已经进行了改善其再生能力。这些的外源运输 因素还越来越关注高成本，药代动力学差和效率低下。这里， 外泌体内内在诱导/治疗分子的增强表现出有希望的治疗性 战略。骨骼细胞分泌重要的生长因子，例如BMP2，这促进了成骨细胞的承诺 骨骼祖细胞和随后的矿物沉积。响应BMP刺激，MSC或 成骨细胞提升了像Noggin这样的BMP拮抗剂，表明有负反馈，以防止过度暴露 BMP信号。揭示了外源性鼻子的引入会损害颅骨形成，同时抑制 内源性鼻子通过激活内源性BMP信号传导促进了颅骨再生。这些 观察结果凸显了抑制内源性BMP活性上调和 随后的骨沉积。在我们的初步研究中，EMS是通过MSC通过独特的 挤出方法，表明具有明显的成骨诱导的外泌体产量很高。而且， 从Noggin抑制MSC（EM-NG）获得的EMS揭示了Noggin siRNA和成骨的升高 效力。我们共同假设EM-NG可以增强内源性的成骨细胞承诺 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点 假设：1）研究EM-NG对内源性颅骨SSC和骨愈合的影响； 2）到 检查载有EM-NG的支架的植入物，以恢复节段下颌缺陷。这 该提案的完成将为进一步开发无单元的方法提供重要的基础 临床颅面缺陷修复。 EMS在颅面恢复中的额外作用将进一步探讨 将来在大型动物模型中。这些未来的研究将在随后的R01应用中提出。