Intraoperative bioprinting of composite tissues with zonal stratification for craniomaxillofacial reconstruction

项目总结/摘要 目前修复颅颌面（CMF）缺损的方法存在一些局限性， CMF缺陷的无缝重建是非常具有挑战性的，因为多个缺陷的精确逐层堆叠， 组织隔室不是微不足道的。这种划分需要精确和有效地利用 干细胞和分化因子，以及将干细胞分化成多个谱系是至关重要的， 重现了天然组织的解剖结构。随着三维（3D）生物打印的进步， 用于CMF修复的原位复合组织最近变得可行，因为3D生物打印能够实现复杂的 以解剖学上准确和美观的方式来观察组织异质性。术中 生物打印，其可以被定义为直接生物打印到缺损区域中以用于修复手术中的损伤 设置，是一个非常有效的过程CMF重建，其中缺陷信息可以迅速 以最少的手动干预获得，能够在手术后立即进行准确的个性化重建， 缺陷的特征。在这个项目中，我们假设术中生物打印的多层 加载有分化因子的复合组织，包括微RNA（miRNA）转染的人 祖细胞和脂肪来源的细胞外基质成分（adECM）诱导细胞间质的区室化， 在无胸腺大鼠模型上概括CMF组织解剖学的软组织和硬组织。为了测试我们的 Specific Aim I将使用术中骨组织生物打印来揭示miR-148 b的影响。 转染的人脂肪源性干细胞（ADSC）对骨组织再生的影响。在 具体目标II，我们将在术中生物打印多层皮肤组织，包括脂肪和真皮层， 为了探索人ADSC和adECM组分的局部递送在不同条件下的影响， 剂量和浓度对皮肤组织再生的影响。特别是，我们将观察ADSC是否 分化成脂肪细胞，并了解脂肪层对真皮的影响 再生在Specific Aim III中，我们将在术中生物打印三层复合组织，包括 颅骨、脂肪层和真皮层，以了解血管化对软组织和硬组织的作用 再生此外，我们还将探讨miR-210在血管形成中的作用。在这方面，我们已经形成了 一个互补的合作，融合了生物打印，再生医学， CMF外科手术、整形外科手术、基因治疗、基因递送、骨力学以及骨和皮肤生物学， 推动拟议工作取得有意义的进展所需的深度，否则这些进展将不会 可能预计拟议工作的成功完成将产生先进的生物打印技术。 这项技术揭示了在一个复杂的组织中工程组织分层之间的复杂相互作用， 免疫缺陷啮齿动物模型，从而提供了对如何局部递送 分化因素影响颅颌面重建。

PROJECT SUMMARY/ABSTRACT Current approaches in repairing craniomaxillofacial (CMF) defects possess several limitations and the reconstruction of CMF defects seamlessly is highly challenging, as precise layer-by-layer stacking of multiple tissue compartments is not trivial. Such compartmentalization necessitates the precision and effective use of stem cells and differentiation factors, and differentiating stem cells into multiple lineages is crucial in order to recapitulate the native tissue anatomy. With the advances in three-dimensional (3D) bioprinting, reconstruction of composite tissues in situ for CMF repair has recently become feasible as 3D bioprinting enables complex tissue heterogeneity in an anatomically accurate and cosmetically appealing manner. Intraoperative bioprinting, which can be defined as bioprinting directly into the defect area for repairing injuries in a surgery setting, is a highly effective process for CMF reconstruction, where the defect information can be rapidly acquired with minimum manual interventions, enabling accurate personalized reconstructions immediately after characterization of the defect. In this project, we hypothesize that intraoperatively bioprinted multi-layer composite tissues loaded with differentiation factors including microRNA(miRNA)-transfected human progenitor cells and adipose-derived extracellular matrix components (adECM) induce compartmentalization of soft and hard tissues that recapitulates CMF tissue anatomy on an athymic rat model. In order to test our hypothesis, Specific Aim I will use intraoperative bone tissue bioprinting to reveal the impact of miR-148b- transfected human adipose-derived stem cells (ADSCs) at respective dosages on bone tissue regeneration. In Specific Aim II, we will intraoperatively bioprint multi-layer skin tissues, including adipose and dermis layers, in order to explore the impact of localized delivery of human ADSCs and adECM components at different dosages and concentrations on skin tissue regeneration, respectively. Particularly, we will observe if ADSCs differentiate into adipocytes and also understand the impact of the presence of adipose layer on dermis regeneration. In Specific Aim III, we will intraoperatively bioprint three-layer composite tissues, including cranium, adipose and dermis layers, in order to understand the role of a vascularization on soft and hard tissue regeneration. In addition, we will explore the role of miR-210 in vascularization. In this regard, we have formed a complementary collaboration that merges essential domain knowledge in bioprinting, regenerative medicine, CMF surgery, plastic surgery, gene therapy, gene delivery, bone mechanics, and bone and skin biology with the depth necessary to propel the proposed work towards meaningful advances that would otherwise not be possible. Successful completion of the proposed work is anticipated to give rise to an advanced bioprinting technology revealing the complex interactions between stratified layers of engineered tissues in an immunodeficient rodent model and thereby provide a novel understanding of how localized delivery of differentiation factors impacts craniomaxillofacial reconstruction.