Small RNA-Modulated Exosome Mimetics For Craniofacial Regeneration

摘要 巨大的颅面缺损仍然是临床外科重建面临的巨大挑战。传统型 自体/同种异体颅面重建的方法受到以下因素的严重影响 可获得性和供体部位发病率。具有多潜能的间充质干细胞(MSCs)日益增多 作为一种基于细胞的骨骼再生方法。尽管如此，累积的证据表明 骨髓间充质干细胞对再生组织的贡献是有限的，而通过 旁分泌起着更重要的作用。间充质干细胞来源的细胞外小泡(EVS)已经显示 在不同动物模型中的再生能力和表现出的治疗优势，如内在归巢 有效、稳定、低免疫原性和有效的信号刺激。然而，广泛使用的 外切体介导的治疗仍然需要显著提高产量和再生能力 才能。开发一种可扩展的方法，如产生外切体模拟(EMS)，并获得可观的产量 已经在以前的研究中进行了调查。此外，Exosome介导的外源性治疗因子的运输 像siRNAs一样，已经进行了提高其再生能力的研究。这些物质的外源运输 因素也越来越受到人们的关注，如成本高、药代动力学差和效率低下。在这里， 在外切体中增强内在的诱导/治疗分子显示出一种很有前途的治疗方法 策略。骨骼细胞分泌重要的生长因子，如BMP2，它刺激成骨细胞承诺 骨骼祖细胞和随后的矿物质沉积。作为对BMP刺激的反应，MSCs或 成骨细胞升高BMP拮抗剂，如noggin，提示负反馈，以防止过度暴露 BMP信令。外源性noggin的引入被发现损害了颅骨的形成，而抑制了 内源性noggin通过激活内源性BMP信号促进颅骨再生。这些 观察强调了noggin抑制内源性BMP活性上调的效力，以及 随后的骨性沉积。在我们的初步研究中，从MSCs通过一种独特的 挤压方法，展示了具有明显成骨诱导作用的外体的高产量。此外， 从noggin抑制MSCs(EM-NG)获得的EMS显示noggin siRNA升高和成骨 威力。综上所述，我们假设EM-NG可以增强成骨细胞对内源性 骨骼干细胞(SSCs)与颅面再生。提出了两个具体的目标来调查这一点 假设：1)研究EM-NG对内源性颅骨SSCs和骨愈合的影响；2)研究EM-NG对SSCs和骨愈合的影响 观察EM-NG复合支架修复下颌骨节段性缺损的效果。这个 这项提议的完成将为进一步开发有效的无细胞方法提供重要的基础 临床颅面缺损修复。EMS在颅面修复中的其他作用将进一步探讨 在未来的大型动物模型中。这些未来的研究将在随后的R01应用中提出。

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.