Harnessing the Power of Apoptosis to Create Regenerative Acellular Biologic Scaffolds

再生医学的许多成功都是通过使用天然来源的材料，特别是脱细胞基质来实现的，例如脱细胞的人真皮和脱细胞的人神经移植物。这些脱细胞基质进一步发展的一个关键限制是，在标准的脱细胞处理过程中，基质微结构和生物活性的丧失，这通常始于诱导细胞坏死（因损伤而死亡）的过程，在整个基质中释放细胞内成分，随后使用刺激性化学物质去除细胞残留物，这也导致不希望的基质破坏和有利生物活性因子的去除。该项目旨在通过诱导细胞凋亡（程序性细胞死亡）而不是坏死来改变脱细胞技术的范式。在细胞凋亡过程中，细胞从基质中分离出来，形成小的凋亡小体，其中含有易于清除的细胞成分。此外，在细胞凋亡过程中，细胞分泌细胞因子，已知有助于剩余的细胞外基质中的组织再生。细胞凋亡技术将用于开发具有保存微结构和生物活性因子的神经和肺组织特异性基质，可与市购产品进行比较。神经组织基质将在大鼠坐骨神经缺损模型中进行测试，并与市售产品进行比较。获得的结果有望通过创建一个新的平台来生成具有增强再生潜力的非细胞组织特异性基质，从而为医学研究界做出贡献，这可能会极大地改变再生医学的未来。通过培训一名研究生和几名本科生，开发有趣的互动课程和实验室，向中学生教授生物材料和生物医学工程，并提高年轻女性对STEM的兴趣，将实现教育和推广影响。该项目的目标是利用细胞凋亡诱导的细胞死亡的力量来创造具有保留微结构和生物活性因子的组织特异性基质，从而增强组织和器官的再生能力。自然来源的材料，特别是脱细胞基质已经成为再生医学成功的关键。进一步发展这些脱细胞基质的一个关键限制是在标准的脱细胞处理过程中失去了精细的基质结构和生物活性。本研究的假设是，通过细胞凋亡诱导细胞死亡将使细胞和抗原的清除更容易、更温和，从而更好地保存基质微结构和生物活性因子。此外，凋亡细胞分泌因子促进移植后的再生。来源于组织特异性细胞外基质的生物支架在组织工程和再生医学中是非常理想的，因为它们由植入部位的天然蛋白质组成，并为再生提供了支架。然而，重要的是在植入前去除所有的细胞成分，以防止免疫原性。最常用的商业脱细胞始于诱导细胞坏死的过程，在整个基质中释放细胞内成分，然后使用刺激性化学物质去除细胞残留物，这也会导致不希望的基质破坏和有益生物活性因子的去除。该项目旨在通过诱导细胞凋亡而不是坏死来改变脱细胞技术的范式。在细胞凋亡过程中，细胞从基质中分离出来，形成含有细胞成分的小凋亡体，这些细胞成分很容易被清除。此外，在细胞凋亡过程中，细胞分泌的细胞因子可以被隔离在ECM中，并且已知可以诱导增殖、干细胞募集和免疫调节，从而帮助组织再生。在Specific Aim中，我利用代表性组织和器官（神经和肺）中的细胞凋亡来创建具有保留微结构和生物活性因子的新型组织特异性基质。将细胞凋亡优化的脱细胞移植物的生物活性和再生潜力与商业上使用的脱细胞技术的移植物进行比较。特异性目标2使用体内模型来分析细胞凋亡优化的脱细胞移植物与商业使用的脱细胞技术产生的移植物的免疫反应和再生潜力。获得的结果有望通过创建一个新的平台来生成具有增强再生潜力的非细胞组织特异性基质，从而为医学研究界做出贡献，这可能会极大地改变再生医学的未来。通过对一名研究生和几名本科生的培训，开发有趣的互动课程和实验室，向中学生教授生物材料和生物医学工程，为Girl？Place, Inc.增加年轻女性人数？他还与盖恩斯维尔的凯德博物馆（Cade Museum of Gainesville）建立了新的合作关系，将课程扩展到全面的实验室，这些实验室将定期运行，以鼓励孩子们的创造力和发明能力。

1605223 - SchmidtMany successes in regenerative medicine have been achieved through the use of naturally derived materials and particularly decellularized matrices, e.g., decellurized human dermis and decellurized human nerve graft. One key limitation to further advancement of these decellularized matrices is a loss of matrix microarchitecture and bioactivity during standard decellularization processing, which often starts with a process to induce cell necrosis (death through damage), releasing intracellular components throughout the matrix, followed by washes using harsh chemicals to remove cell remnants, which also results in undesired matrix destruction and removal of favorable bioactive factors. This project seeks to shift the paradigm in decellularization techniques by inducing apoptosis (programmed cell death) rather than necrosis. During apoptosis cells detach from the matrix and form small apoptotic bodies containing cellular components that can be readily removed. Furthermore, during apoptosis cells secrete cytokines that are known to aid in tissue regeneration in the remaining extracellular matrix. The apoptosis technique will be used to develop nerve and lung tissue specific matrices with preserved microarchitecture and bioactive factors that can be compared to commercially available products. The nerve tissue matrix will be tested in a rat sciatic nerve defect model, also in comparison with commercially available products. Results obtained are expected to contribute to the medical research community by creating a new platform for generation of acellular tissue specific matrices with enhanced regenerative potential, which could dramatically shift the future of regenerative medicine. Educational and outreach impact will be achieved through training of a graduate student and several undergraduate students and developing fun interactive lessons and labs to teach middle school students about biomaterials and biomedical engineering and to increase young women's interest in STEM.The goal of this project is to harness the power of apoptosis induced cell death to create tissue specific matrices with preserved microarchitecture and bioactive factors capable of enhancing regeneration in tissues and organs. Naturally derived materials and particularly decellularized matrices have been essential to success in regenerative medicine. One key limitation to further advancement of these decellularized matrices is a loss of fine matrix structure and bioactivity during standard decellularization processing. The hypothesis of this work is that induction of cell death by apoptosis will enable easier and gentler cell and antigen removal, thus better preserving matrix microarchitecture and bioactive factors. Furthermore, apoptotic cells secrete factors to enhance regeneration after implantation. Biological scaffolds derived from tissue specific extracellular matrix are highly desirable for tissue engineering and regenerative medicine because they are composed of proteins native to the implant site and provide a scaffold for regeneration. However, it is important to remove all cellular components prior to implantation to prevent immunogenicity. Most commonly used commercial decellularization start with a process to induce cell necrosis, releasing intracellular components throughout the matrix, followed by washes using harsh chemicals to remove cell remnants, which also results in undesired matrix destruction and removal of favorable bioactive factors. This project seeks to shift the paradigm in decellularization techniques by inducing apoptosis rather than necrosis. During apoptosis cells detach from the matrix and form small apoptotic bodies containing cellular components, which can be readily removed. In addition, during apoptosis cells secrete cytokines that can be sequestered in the ECM, and are known to induce proliferation, stem cell recruitment, and immunomodulation, thereby aiding in tissue regeneration. In Specific Aim 1 exploits cell apoptosis in representative tissues and organs (nerve and lung) to create novel tissue specific matrices with preserved microarchitecture and bioactive factors. Bioactivity and regenerative potential of apoptosis-optimized acellular grafts will be compared to grafts from a commercially used decellularization technique. Specific Aim 2 uses in vivo models to analyze immune response and regenerative potential of apoptosis-optimized acellular grafts versus grafts created from a commercially used decellularization technique. Results obtained are expected to contribute to the medical research community by creating a new platform for generation of acellular tissue specific matrices with enhanced regenerative potential, which could dramatically shift the future of regenerative medicine. Educational and outreach impact will be achieved through training of a graduate student and several undergraduate students, developing fun interactive lessons and labs to teach middle school students about biomaterials and biomedical engineering, developing lessons for 6th-8th grade girls at Girl?s Place, Inc. to increase young women?s interest in STEM and forging a new collaboration with the Cade Museum of Gainesville to expand lessons into full scale labs that will be run regularly to encourage creativity and invention in children.