Cell delivery for irradiated bone defects

描述(申请人提供)：受辐射患者的骨骼愈合受损是由于局部细胞群受损和血管系统受损所致。基于细胞的治疗是组织移植的一种强有力的替代方案，但其临床应用需要容易获得的、具有血管生成潜力的人类细胞，并且在适当的刺激下有助于骨形成。内皮细胞集落形成细胞(ECFC)是一类在低氧条件下表现出强大的血管生成能力的内皮祖细胞亚群，在体内可以形成功能性的血管网络。间充质干细胞(MSCs)具有易回收、增殖快、具有成骨潜能、支持毛细血管形成等特点。成功的以细胞为基础的骨愈合疗法将通过利用能够协同促进组织修复的群体来确定，当与将移植群体定位到缺损处的基质一起输送时，同时还促进和增强细胞的存活、增殖和分化。纤维蛋白凝胶模拟血液凝块，这是自然骨折部位愈合的第一步，之所以吸引人，是因为它们是可生物吸收的、可注射的，纤维蛋白可以从患者自己的血液中制备，就像天然的血液凝块一样，它们有效地阻止了对愈合至关重要的细胞。纤维蛋白凝胶的化学和机械性能可以通过改变凝血蛋白的相对浓度来调整。更坚硬的基质通过干细胞和祖细胞驱动骨形成。作为增加纤维蛋白凝胶硬度所必需的高凝蛋白浓度的替代品，我们最近开发了一种通过操纵离子强度来增加纤维蛋白凝胶硬度的方法。我们的中心假设是，物理性能增强的纤维蛋白凝胶将成为共移植直接参与新生血管和骨形成的细胞群体的有效载体。目的1.测定内皮细胞和骨髓间充质干细胞在纤维蛋白凝胶中共培养的血管生成和成骨潜能。工程纤维蛋白凝胶支持被包裹的细胞的血管生成和成骨反应的能力将被量化。评估共移植于纤维蛋白凝胶内的ECFCs和MSCs在照射后的啮齿类动物颅骨缺损模型中促进血管形成和骨形成的能力。植入的细胞对组织灌流和骨形成的贡献将使用非侵入性成像方式进行评估。这项拟议的研究具有创新性，因为它利用了组织愈合、纤维蛋白和组织形成细胞的自然参与者，以克服抑制正常修复的组织微环境。我们将阐明ECFCs和MSCs在植入具有头颈癌患者特征的放射性骨缺损中的作用。总而言之，这项研究将填补我们在理解血管和基质细胞如何促进毛细血管形成和组织修复方面的关键空白，这些研究对治疗不可愈合的骨缺损、软组织缺损以及组织工程和再生医学中的新兴应用具有直接意义。 公共卫生相关性：开发新的细胞递送策略，在植入后加速组织形成和功能，将极大地提高那些患有不可愈合组织缺损者的生活质量。我们试图确定具有更高硬度的工程纤维蛋白凝胶是否可以通过移植血管形成细胞和骨形成细胞来促进血管形成和骨形成。

DESCRIPTION (provided by applicant): Impaired bone healing in irradiated patients results from damage to local cell populations and a compromised vasculature. Cell-based therapies represent a powerful alternative to tissue grafts, yet their clinical application requires human cells that can be readily obtained, possess angiogenic potential, and contribute to bone formation upon appropriate stimulation. Endothelial colony forming cells (ECFCs) are a subpopulation of endothelial progenitor cells that exhibit robust angiogenic potential under hypoxic conditions and can form functional vascular networks in vivo. Mesenchymal stem cells (MSCs) are easily retrievable and proliferate rapidly, possess osteogenic potential, and support capillary formation. Successful cell-based therapies for bone healing will be identified by utilizing populations that can synergistically contribute to tissue repair when delivered with a matrix that localizes transplanted populations to the defect site, while also facilitating and enhancing cell survival, proliferation, and differentiation. Fibrin gels mimic the blood clot that is the first step of healing in a natural fracture site and are appealing because they are bioresorbable, injectable, the fibrin can be prepared from the patient's own blood and, like a natural blood clot, they effectively hold cells critical to healing. Chemical and mechanical properties of fibrin gels may be tailored by modifying the relative concentrations of clotting proteins. Stiffer substrates drive bone formation with stem and progenitor cells. As an alternative to high clotting protein concentrations necessary to increase fibrin gel stiffness, we recently developed a method for increasing the stiffness of fibrin gels by manipulating ionic strength. Our central hypothesis is that fibrin gels with enhanced physical properties will be effective vehicles for cotransplanting cell populations that directly participate in neovascularization and bone formation. Aim 1. Determine the vasculogenic and osteogenic potential of ECFCs and MSCs co-cultured within fibrin gels possessing increased stiffness. The ability of engineered fibrin gels to support the vasculogenic and osteogenic response of entrapped cells will be quantified. Assess the ability of ECFCs and MSCs cotransplanted within fibrin gels to enhance vascularization and bone formation in an irradiated rodent calvarial defect model. The contribution of implanted cells toward tissue perfusion and bone formation will be assessed using noninvasive imaging modalities. The proposed research is innovative because it exploits natural participants of tissue healing, fibrin and tissue-forming cells, to overcome tissue microenvironments that inhibit normal repair. We will elucidate the contributions of ECFCs and MSCs when implanted into an irradiated bone defects characteristic of patients with head and neck cancer. Collectively, this research will fill a critical void in our understanding of how vascular and stromal cells promote capillary vessel formation and tissue repair, and these studies have direct relevance for treating nonhealing bone defects, soft tissue defects, and emerging applications in tissue engineering and regenerative medicine. PUBLIC HEALTH RELEVANCE: The development of new cell delivery strategies that accelerate tissue formation and function upon implantation will greatly improve the quality of life for those who suffer from nonhealing tissue defects. We seek to determine if engineered fibrin gels possessing increased stiffness can promote vascularization and bone formation by transplanted vessel- and bone-forming cells.