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

描述（由申请人提供）：大量节段性骨丢失后的肢体抢救是军事和民用骨科领域的主要挑战。严重的骨缺损手术需要大量失活的异体骨段移植来替代缺失的宿主骨段，然而由于失活的异体骨段融入宿主骨的能力受损，经常出现严重的问题。促进和增强同种异体移植结合和关键骨缺损愈合的最令人兴奋的策略之一涉及使用患者自身骨髓来源的间充质基质/祖细胞（MSCs）。虽然这种方法已经取得了一些临床前的成功，但在大多数情况下仍然存在一些问题，包括：通过培养技术，间充质干细胞与同种异体移植物的附着效率低下，间充质干细胞在移植物中的分布不均匀，以及间充质干细胞与移植物的粘附力弱，导致体内细胞脱落。为了克服这些问题，生物可降解聚合物支架已被广泛应用于细胞制造三维组织样移植物。然而，在支架的生物降解过程中经常观察到强烈的炎症反应。因此，一种避免使用生物可降解支架的新方法被强烈期望用于同种异体移植。利用温度响应培养皿的细胞片技术已经在组织工程中应用了好几年，用于再生受损组织。通过这项技术，与标准细胞培养皿中产生的细胞片相比，细胞片可以很容易地从培养皿中分离出来，没有任何表面蛋白质和细胞外基质（ECM）损伤，并作为组织工程骨膜移植到大骨缺损部位。与直接植入MSCs的同种异体移植物相比，我们预计这种新技术将显著加速同种异体移植物与宿主骨的结合。最近，我们已经成功地分离和培养了人骨髓来源的间充质干细胞。在本应用中，我们将描述由细胞片培养技术引起的表型变化，并确定使用从患者身上新分离的人间充质干细胞（通道3）和广泛传代的间充质干细胞（通道10）生成间充质干细胞片所需的最佳细胞培养条件。其次，我们将测试人类间充质干细胞片修复免疫缺陷小鼠临界大小骨缺损的能力。该提案产生的数据可能会确定一种新的方法来产生组织工程骨膜，以使用于关键节段性骨缺损手术的大量失活同种异体移植物恢复活力，这将最终改善对大骨缺损或其他组织损伤患者的护理和管理。

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.