Genetically Designed Materials for Cartilage Repair

描述（由申请人提供）：健康椎间盘功能的维持取决于髓核（NP）解剖结构完整性和功能的保留。与衰老相关的细胞损失使椎间盘几乎没有能力对损伤或与衰老相关的变化做出反应，例如椎间盘高度或水合作用的损失、椎间盘裂隙或挤压，所有这些特征都与症状性椎间盘疾病相关。现在，使用自体、祖细胞和其他携带体内再生NP样基质潜力的细胞补充椎间盘引起了极大的兴趣。然而，再生NP样基质所需的独特和特定的细胞特征知之甚少，并且很少有技术策略可用于体外或体内帮助实现这一目标。我们已经确定了未成熟的NP细胞的独特功能是他们的粘附特定的层粘连蛋白，层粘连蛋白结合受体和层粘连蛋白相关蛋白的表达。这些独特的特征已经在三个物种（猪、大鼠和人NP）中鉴定，并且包括结合层粘连蛋白-1和层粘连蛋白-10、表达整联蛋白和非整联蛋白层粘连蛋白受体以及表达层粘连蛋白相关蛋白的能力。此外，层粘连蛋白结合保护细胞免于血清剥夺诱导的细胞凋亡，并促进独特地含有层粘连蛋白和这些相关受体的细胞外基质的形成。我们建议合成新的生物材料，促进层粘连蛋白结合原代NP细胞和成人祖细胞（hADAS），以刺激再生的一个独特的，NP样细胞外基质。在目标1中，我们将选择人类和猪NP细胞群体，用于它们与层粘连蛋白或细胞结合层粘连蛋白肽的附着，并将它们封装在由层粘连蛋白和弹性蛋白样多肽（ELP）组成的3D多肽水凝胶中。将研究新合成的基质的特异性层粘连蛋白相关和其他NP基质标志物的表达。还将研究细胞附着强度和受体表达。将使用人工神经网络对细胞群之间基质的数量或质量、附着强度和受体表达谱的差异进行建模，并进行测试以确定层粘连蛋白结合是否促进增强的再生NP的能力。在目标2中，我们将设计作为3D支架的细胞结合层粘连蛋白肽和ELP的融合蛋白。提出了保留3D水凝胶形成ELP结构域，以及引入NP细胞特有的细胞结合结构域，以促进NP样基质的再生。这项工作的意义将是一种新的细胞指导性生物材料的设计，结合一个容易获得的细胞来源，可以作为一个有效的，广泛可用的治疗选择NP样基质再生的病理性NP变化的治疗。

DESCRIPTION (provided by applicant): Maintenance of healthy intervertebral disc function depends on preservation of the anatomic integrity and function of the nucleus pulposus (NP). Aging-related cell losses leave the disc with little ability to respond to injury or aging-related changes such as loss of disc height or hydration, disc fissures or extrusion, all features associated with symptomatic intervertebral disc disorders. Cell supplementation to the disc is now of great interest, using autologous, progenitor and other cells that carry the potential to regenerate NP-like matrix in vivo. Little is known of unique and specific cellular characteristics required for regenerating NP-like matrix, however, and there are few technical strategies available to assist this goal in vitro or in vivo. We have identified unique features of immature NP cells to be their adhesion to specific laminins, and their expression of laminin-binding receptors and laminin-associated proteins. These unique features have been identified in three species (porcine, rat and human NP) and include an ability to bind to laminin-1 and laminin-10, to express both integrin and non-integrin laminin receptors, and to express a laminin associated protein. Furthermore, laminin binding protects cells from serum deprivation-induced cell apoptosis and promotes formation of extracellular matrix that uniquely contains laminin and these associated receptors. We propose to synthesize new biomaterials that promote laminin binding for primary NP cells and adult progenitor (hADAS) cells, in order to stimulate regeneration of a distinct, NP-like extracellular matrix. In Aim 1, we will select human and porcine NP cell populations for their attachment to laminin or cell-binding laminin peptides and encapsulate them in 3D polypeptide hydrogels composed of laminins and elastin-like polypeptides (ELPs). Newly synthesized matrix will be studied for its expression of specific laminin-associated and other NP matrix markers. Cell attachment strength and receptor expression will also be studied. Differences in the quantity or quality of matrix, attachment strength and receptor expression profile, amongst cell populations will be modeled using artificial neural networks and tested to determine if laminin binding promotes an enhanced capacity to regenerate the NP. In Aim 2, we will design fusion proteins of cell-binding laminin peptides and ELPs that function as 3D scaffolds. Preservation of the 3D hydrogel-forming ELP domain, together with introduction of a cell- binding domain unique to NP cells, is proposed to promote the regeneration of NP-like matrix. The significance of this work will be the design of a new cell-instructive biomaterial that, combined with a readily accessible cell source, can serve as an effective, broadly available therapeutic option for NP-like matrix regeneration for the treatment of pathological NP changes.