Collaborative Research: 3D-Biofabrication of hASC-based Biomimetic Osteochondral Tissue and the Role of Extracellular Calcium Receptor

骨关节炎（osteoarthritis， OA）是一种退行性关节疾病，由于软骨逐渐退化，限制了受影响关节的活动。目前，美国有超过2500万人患有这种疾病，而且预计这个数字只会上升。目前软骨和骨软骨组织修复的临床方法受到供体部位发病率、组织稀缺性和长期功能预后差的限制。如果工程组织能够模仿天然组织的特征，那么它们可能会成为更有效的替代品。本项目研究了一种新的骨软骨组织工程策略，使用人类脂肪来源（脂肪来源）干细胞（hASCs）和3D生物制造技术。3D结构将被设计成模拟天然骨软骨组织的结构和生物学特征。这种结构将使用受生理启发的材料进行生物3d打印，将产生特定部位的hASCs软骨形成和成骨。细胞外钙敏感受体在调节hASC分化中的机制作用，对这种工程构建至关重要的控制，也将被研究。工程结构的特性将使用体外方法进行评估，并使用微型猪模型进行概念验证研究。创造这种基于hasc的仿生组织移植的能力可以导致骨关节炎治疗的重要临床进展。钙感应受体（CaR）如何影响干细胞分化的知识可以用于开发其他肌肉骨骼组织和再生医学应用的工程组织替代品。该项目还将直接支持合作院校的博士后学者、研究生和本科生的多学科教育。pi将继续关注和努力招收STEM中代表性不足的群体的学生。研究的学习和成果将被纳入多个本科和研究生课程，并在包括大学开放日和夏令营在内的外展活动中使用。重要的研究结果将通过学术期刊出版物以及在地方、国家和国际会议上传播。该项目的重点是研究一种新的3D组织工程方法，可能转化为OA治疗。具体地说,一种新型的生物3D打印四相结构将使用生理启发的化学线索来诱导人类脂肪来源的干细胞的位点特异性分化，其方式概括了骨软骨组织的深度依赖成分和结构异质性，从骨软骨组织的致密表层到多孔软骨下的骨软骨组织的深度依赖成分和结构异质性。这对它在承载过程中的功能至关重要。细胞外钙敏感受体在调节hASC成骨和软骨谱系规范中的机制作用，其控制对这种工程构建至关重要，也正在研究中。为了使基础科学的进步，同时确保未来的临床相关性，将使用体外方法和体内模型的协同方法来实现项目目标。主要假设是，生物打印的β -三磷酸钙（TCP）和具有优化立孔结构的脱细胞关节软骨细胞外基质（dECM）水凝胶的梯度将诱导位点特异性hASC成骨和软骨形成，其中CaR在这一谱系规范中发挥核心作用。这一假设将通过完成以下具体目标来验证：1)研究和表征TCP和软骨dECM水凝胶梯度（以聚已内酯（PCL）为基础的复合材料）及其3d生物标记的链孔几何形状对hASC的部位特异性成骨和软骨形成的影响，2)确定CaR的失活（siRNA和MPS2143， calytic）和激活（Cinacalcet, calimimetic）对hASC对细胞外Ca2+的反应的影响。特别是，评估CaR调节hASC成骨和软骨形成的能力。3)利用猪骨软骨缺损模型和人类手术中使用的手术方法和工具，评估体内部位特异性hASC成骨和软骨分化和优化的全层四相结构的整合（概念验证试点研究）。计划的方法克服了当前临床方法的许多局限性，即无法模仿原生组织异质性。该项目采用多学科方法，从制造，生物医学工程和临床科学，以发展细胞外Ca2+和CaR在hASC分化中的作用的理解，并代表了一个新的方法来创建多相仿生组织具有临床翻译的潜力。从组织设计和制造的角度来看，该项目将为PCL-TCP/dECM复合结构的3D生物绘图提供关于材料-过程-结构相互作用的新知识。从临床科学的角度来看，理解和阐明CaR在调节细胞外Ca2+对hASC谱系规范的影响中的作用将导致对CaR活化的药理学抑制如何用于优化基于hASC的方法来设计所需组织界面（例如软骨，韧带）的骨和/或软骨梯度的新理解。使用hASC成功的骨软骨TE将允许利用患者丰富的ASC进行自体组织移植。hasc诱导移植复制全层骨软骨组织的能力将是软骨和骨软骨缺损修复的重要临床进展。了解CaR在hASC谱系规范中的机制作用将为hASC在其他肌肉骨骼TE和再生医学应用中的应用提供关键知识。

PIs: Loboa, Elizabeth/Shirwaiker, RohanProposals: 1702841/1703466Osteoarthritis (OA)is a degenerative joint disease that limits mobility of the affected joint due to gradual degradation of the cartilage. It currently affects over 25 million people in the US, and these numbers are only expected to rise. Current clinical approaches to cartilage and osteochondral tissue repair are limited by donor site morbidity, tissue scarcity, and poor long term functional outcomes. Engineered tissues can potentially serve as more effective alternatives if they can be designed to mimic native tissue characteristics. This project investigates a new osteochondral tissue engineering strategy using human adipose-derived (fat-derived) stem cells (hASCs) and 3D biofabrication techniques. A 3D construct will be engineered to mimic the structural and biological characteristics of native osteochondral tissue. This construct will be 3D-bioprinted with physiologically-inspired materials that will produce site-specific chondrogenesis and osteogenesis of hASCs. The mechanistic role of the extracellular calcium sensing receptor in regulating hASC differentiation, control that is critical for such an engineered construct, will be also be studied. The characteristics of the engineered construct will be assessed using in vitro methods as well as a proof-of-concept study using a miniature pig model. The ability to create such a hASC-based biomimetic tissue transplant can lead to a paramount clinical advancement for osteoarthritis treatment. The knowledge about how the calcium sensing receptor(CaR)influences stem cell differentiation can be utilized to develop engineered tissue alternatives for other musculoskeletal tissues and regenerative medicine applications. This project will also directly support the multidisciplinary education of a post-doctoral scholar, and graduate and undergraduate students at the partnering institutions. The PIs will continue their focus and efforts in recruiting students from groups underrepresented in STEM. The learnings and outcomes from the studies will be incorporated in multiple undergraduate and graduate courses, and used during outreach activities including university open houses and summer camps. Important findings will be disseminated through scholarly journal publications and at local, national and international meetings.The project focuses on investigating a new 3D tissue engineering approach with possible translation to OA treatment. Specifically, a novel 3D bioprinted quadriphasic construct will be engineered using physiologically inspired chemical cues to induce site-specific differentiation of human adipose-derived stem cells in a manner that recapitulates the depth dependent compositional and architectural heterogeneity of osteochondral tissue varying from the depth dependent compositional and architectural heterogeneity of osteochondral tissue varying from the dense surface layer of cartilage to the porous subchondral one, which is crucial to its function during load bearing. The mechanistic role of the extracellular calcium sensing receptor in regulating the osteogenic and chondrogenic lineage specification of hASC, controlling of which is critical for this engineered construct is also being investigated. To enable the advancement of fundamental science while ensuring future clinical relevance, a synergistic approach utilizing in vitro methods and an in vivo model will be used to achieve project goals. The primary hypothesis is that gradients of the bioprinted beta tricalcium phosphate (TCP) and decellularized articular cartilage extracellular matrix (dECM) hydrogel with optimized stand-pore architecture will induce site-specific hASC osteogenesis and chondrogenisis, with the CaR playing a central role in this lineage specification. This hypothesis will be tested by completing the following specific aims: 1) Investigate and characterize the effects of TCP and cartilage dECM hydrogel gradients (in polycaprolactone (PCL) based composites) and their 3D-bioplotted strand-pore geometries on site specific osteogenesis and chondrogenesis, respectively, of hASC, 2) Determine the effects of inactivation (siRNA and MPS2143, calcilytic) and activation (Cinacalcet, calcimimetic) of the CaR on hASC response to extracellular Ca2+. In particular, evaluate the ability of the CaR to regulate hASC osteogenesis as well as chondrogenesis, 3) Assess in vivo site-specific hASC osteo- and chondrogenic differentiation and integration of the optimized full-thickness quadriphasic construct using a porcine osteochondral defect model and surgical approaches and tools used in human surgeries (proof-of-concept pilot study). The planned approach overcomes many of the limitations of current clinical approaches that fail to mimic native tissue heterogeneity. The project uses multidisciplinary methods from manufacturing, biomedical engineering and clinical sciences to develop an understanding of the role of extracellular Ca2+ and the CaR in the differentiation of hASC, and represents a new approach to creating a multiphasic biomimetic tissue with the potential for clinical translation. From a tissue design and manufacturing perspective, the project will provide new knowledge about the material-process-structure interactions in the 3D bioplottig of PCL-TCP/dECM composite constructs. From a clinical science perspective, understanding and elucidating the role of the CaR in regulation of the effects of extracellular Ca2+ on hASC lineage specification will lead to new understanding of how pharmacological inhibition of activation of the CaR could be used to optimize hASC based approaches to engineer bone and/or cartilage gradients at desired tissue interfaces (e.g. cartilage, ligament.). Successful osteochondral TE using hASC will allow for autologous tissue transplantation using a patient's abundant ASC. The ability to place an hASC-induced transplant that replicates full thickness osteochondral tissue will be a paramount clinical advancement for chondral and osteochondral defect repair. Understanding the mechanistic role of the CaR in hASC lineage specification will provide critical knowledge for utilization of hASC in other musculoskeletal TE and regenerative medicine applications.