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保肢是军事和民用领域骨科领域的主要挑战。Critica骨缺损手术需要大的失活节段同种异体移植物移植来替换缺失的宿主骨段，然而，由于失活同种异体移植物结合到宿主骨中的能力受损，通常会出现重大问题。促进和增强同种异体移植物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的骨髓源性间充质基质/祖细胞（MSC）。虽然这种方法已经证明了一些临床前的成功，但在大多数情况下仍然存在问题，包括：通过培养技术将MSC附着到同种异体移植物上的效率低下，MSC在移植物上的分布不均匀，以及MSC与移植物的弱粘附导致体内细胞脱离。为了克服这些问题中的一些，生物可降解聚合物支架已被广泛用于与细胞一起制造三维组织样移植物。然而，在支架生物降解后经常观察到强烈的炎症反应。因此，一种新的方法，以避免使用可生物降解的支架是强烈期待同种异体移植。利用温度响应培养皿的细胞片层技术已经应用于组织工程几年，以再生受损组织。通过这种技术，与标准细胞培养皿中产生的细胞片相比，细胞片可以容易地从培养皿中分离而没有任何表面蛋白和细胞外基质（ECM）损伤，并移植到大的骨缺损部位，作为植入移植物周围的组织工程骨膜。我们预计，这种新技术将显着加速同种异体骨纳入宿主骨相比，使用同种异体骨移植，骨髓间充质干细胞直接接种在移植物。最近，我们已经能够成功地分离和培养人骨髓源性MSC。在本申请中，我们将表征由细胞片培养技术引起的表型变化，并鉴定使用来自患者的新鲜分离的人MSC和传代MSC（Lonza Inc.）产生MSC片所需的最佳细胞培养条件。其次，我们将测试人MSC片层修复免疫缺陷小鼠中临界尺寸骨缺损的能力。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，以振兴用于关键节段性骨缺损手术的大规模失活同种异体移植物，最终改善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 from patients and passaged MSCs (Lonza Inc.). 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.