Genetically Designed Materials for Cartilage Repair

描述（由申请人提供）：维持健康的椎间盘功能取决于保留髓核（NP）的解剖完整性和功能。与衰老相关的细胞损失使椎间盘对损伤或与衰老相关的变化（如椎间盘高度或水合作用丧失、椎间盘裂或挤压）反应能力减弱，所有这些特征都与症状性椎间盘疾病相关。利用自体细胞、祖细胞和其他具有在体内再生np样基质潜能的细胞向椎间盘补充细胞是目前研究的热点。然而，人们对再生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以在体外或体内帮助实现这一目标。我们已经确定了未成熟NP细胞的独特特征，即它们对特定层粘连蛋白的粘附，以及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、大鼠和人类NP）中被发现，包括与层粘连蛋白-1和层粘连蛋白-10结合的能力，表达整合素和非整合素层粘连蛋白受体的能力，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受血清剥夺诱导的细胞凋亡，并促进细胞外基质的形成，细胞外基质独特地含有层粘连蛋白和这些相关受体。我们建议合成新的生物材料，促进层粘胶蛋白结合原代NP细胞和成人祖细胞（hADAS），以刺激独特的NP样细胞外基质的再生。在Aim 1中，我们将选择人类和猪NP细胞群，因为它们附着在层粘连蛋白或细胞结合层粘连蛋白肽上，并将它们包裹在由层粘连蛋白和弹性蛋白样多肽（ELPs）组成的3D多肽水凝胶中。新合成的基质将研究其特异性层粘连蛋白相关和其他NP基质标记的表达。细胞附着强度和受体表达也将被研究。细胞群中基质的数量或质量、附着强度和受体表达谱的差异将使用人工神经网络进行建模，并进行测试，以确定层粘连蛋白结合是否促进了NP再生能力的增强。在Aim 2中，我们将设计细胞结合层粘连蛋白肽和elp的融合蛋白，作为3D支架。提出保留3D水凝胶形成的ELP结构域，并引入NP细胞特有的细胞结合结构域，以促进NP样基质的再生。这项工作的意义在于设计一种新的细胞指导生物材料，与易于获取的细胞来源相结合，可以作为一种有效的、广泛可用的NP样基质再生治疗病理性NP改变的治疗选择。

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.