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

描述(由申请人提供)：在大量节段性骨丢失后的肢体保全是在军事和民用领域对骨科领域的一大挑战。眼球结膜骨缺损术需要大量失活的同种异体骨移植来替代缺失的宿主骨段，然而，由于失活的同种异体骨结合到宿主骨中的能力受损，经常会出现严重的问题。促进和加强同种异体骨的融合和关键的骨缺损愈合的最令人兴奋的策略之一是使用患者自己的骨髓来源的间充质基质/祖细胞(MSCs)。虽然这种方法在临床前取得了一些成功，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效率低下，MSC在移植物上的分布不均匀，以及MSCs在移植物上的粘附力较弱，导致体内细胞分离。为了克服其中的一些问题，可生物降解的聚合物支架已被广泛用于细胞构建三维组织状移植物。然而，在支架的生物降解过程中，经常会观察到强烈的炎症反应。因此，一种避免使用可生物降解支架的新方法在同种异体移植中是非常有希望的。利用温度响应型培养皿的细胞片技术已经应用于组织工程几年，以再生受损组织。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，而不会造成任何表面蛋白和细胞外基质(ECM)损伤，并作为组织工程骨膜移植到植入物周围的大型骨缺损处。我们预计，与将MSCs直接种植在移植物上的同种异体移植物相比，这项新技术将显着加快移植物与宿主骨的结合。最近，我们成功地分离和培养了人骨髓间充质干细胞。在这项应用中，我们将表征细胞片培养技术引起的表型变化，并确定使用患者新鲜分离的人MSCs(第3代)和广泛传代的MSCs(第10代)生成MSC片所需的最佳细胞培养条件。其次，我们将测试人间充质干细胞片修复免疫缺陷小鼠严重骨缺损的能力。这项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，以重振关键节段性骨缺损手术中使用的大量失活同种异体移植物，最终将改善对大型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.