Assessment of stromal cell sheets for repair of critical segmented bone defects

评估基质细胞片修复关键分段骨缺损的效果

• 批准号: 8581667
• 负责人: Yufeng Dong
• 金额: $ 19.57万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581667
• 关键词: Adhesions; Affect; Allografting; Aspirate substance; Biodegradation; Biomechanics; Bone Marrow; Cell Count; Cell Culture Techniques; Cell Nucleus; Cell Size; Cell-Matrix Junction; Cells; Culture Techniques; Data; Defect; Extracellular Matrix; Femur; Flow Cytometry; Future; Gene Expression; Healed; Hour; Human; Immunohistochemistry; Implant; In Situ Hybridization; Inflammatory Response; Lead; Limb Salvage; Membrane Proteins; Mesenchymal; Methods; Military Personnel; Modeling; Mus; Natural regeneration; Nude Mice; Operative Surgical Procedures; Orthopedics; Patient Care Management; Patients; Periosteum; Process; Research; Site; Staining method; Stains; Stem cells; Stromal Cells; Techniques; Technology; Temperature; Testing; Time; Tissue Engineering; Tissues; Transplantation; allogenic bone transplantation; base; biodegradable polymer; bone; bone lead; bone loss; clinical practice; healing; in vivo; innovation; molecular phenotype; mouse model; new technology; novel strategies; novel therapeutic intervention; pre-clinical; public health relevance; reconstruction; regenerative; repaired; response; scaffold; segregation; stromal progenitor; success; tissue regeneration

DESCRIPTION (provided by applicant): Limb salvage following massive segmental bone loss is a major challenge to the field of orthopaedics in both the military and civilian arenas. Critica bone defect surgeries require large devitalized segmental allograft transplantations to replace missing host bone segments, however significant problems often arise due to the impaired ability of the devitalized allograft to incorporate into the host bone. One of the most exciting strategies to promote and enhance allograft incorporation and critical bone defect healing involves the use of the patient's own bone marrow derived mesenchymal stromal/progenitor cells (MSCs). While this approach has demonstrated some preclinical success, problems remain in most cases including: inefficient MSC attachment to allograft via culturing techniques, uneven MSC distribution across the graft, as well as, weak adhesion of MSCs to the graft resulting in cell detachment in vivo. To overcome some of these problems, biodegradable polymer scaffolds have been widely used with cells to fabricate three-dimensional tissue-like grafts. However, strong inflammatory responses are often observed upon biodegradation of the scaffolds. Therefore, a new method to avoid the use of biodegradable scaffolds is strongly expected for allograft transplantation. Cell sheet technology utilizing temperature-responsive culture dishes has been applied to tissue engineering for several years to regenerate damaged tissues. Via this technology, cell sheets can be easily detached from culture dishes without any surface proteins and extracellular matrix (ECM) damage compared to cell sheets generated in standard cell culture dishes, and transplanted to the site of large bone defects, as a tissue-engineered periosteum surrounding the implanted graft. We expect that this new technique will significantly accelerate allograft incorporation into the host bone when compared to the use of allografts that have had MSCs directly seeded on the graft. Recently, we have successfully been able to isolate and culture human bone marrow derived MSCs. In this application, we will characterize the phenotypic changes caused by the cell sheet culturing techniques, and identify the optimal cell culture conditions necessary to generate MSC sheets using both freshly isolated human MSCs (passage3) from patients and extensively passaged MSCs (passage10). Secondly, we will test the ability of human MSC sheets to repair a critical sized bone defect in the immuno- deficient mouse. The data generated by this proposal will likely identify a novel approach to generate a tissue- engineered periosteum to revitalize massive devitalized allografts used in critical segmented bone defect surgeries that will ultimately lead to improvements in the care and management of patients with large bone defects or other tissue damage.

描述（由申请人提供）：大规模分段骨质流失后的肢体打捞是军事和平民竞技场中骨科领域的主要挑战。批评的骨缺陷手术需要大量退化的分段同种异体移植移植才能替代缺失的宿主骨骼片段，但是由于远离同种异体移植物将其掺入宿主骨骼的能力受损，经常出现重大问题。促进和增强同种异体移植掺入和临界骨缺损愈合的最令人兴奋的策略之一是使用患者自身的骨髓衍生的间充质基质/祖细胞（MSC）。尽管这种方法已显示出一些临床前的成功，但在大多数情况下仍然存在问题：包括：通过培养技术效率低下的MSC附着在同种异体移植物上，移植物的MSC分布不均匀，以及MSC对移植物的粘附较弱，导致体内细胞脱离。为了克服其中一些问题，可生物降解的聚合物支架已与细胞广泛使用，以制造三维组织样的移植物。但是，经常在脚手架的生物降解后观察到强烈的炎症反应。因此，强烈期望一种避免使用可生物降解支架的新方法用于同种异体移植。使用温度响应性培养皿的细胞表技术已应用于组织工程已有数年的时间来再生受损的组织。通过这项技术，与在标准细胞培养皿中产生的细胞表相比，可以轻松地将细胞表从没有表面蛋白和细胞外基质（ECM）损伤的培养皿中分离出来，并将其移植到大骨缺陷的部位，作为植入植入植物周围的组织骨膜。我们预计，与使用直接在移植物上接种MSC的同种异体移植物相比，这种新技术将显着加速同种异体移植。最近，我们成功地孤立了人类骨髓衍生的MSC。在此应用中，我们将表征由细胞表培养技术引起的表型变化，并确定使用患者和广泛传递的MSC（Passage10）使用新鲜分离的人MSC（Passage3）生成MSC表所需的最佳细胞培养条件。其次，我们将测试人类MSC板在免疫缺陷小鼠中修复临界大小的骨缺损的能力。该提案产生的数据可能会确定一种新的方法来产生组织设计的骨膜，以振兴在关键的分段骨缺损手术中使用的大规模退化化同种异体移植物，最终会改善较大骨缺陷或其他组织损害的患者的护理和管理。