Engineering Microenvironments for the Nucleus Pulposus Cell

 描述(申请人提供)：椎间盘的髓核(NP)细胞来自脊索，合成一种柔软的凝胶基质，含有大量的胶原、蛋白多糖和层粘连蛋白。随着年龄的增长，NP细胞变得更像成纤维细胞，并失去合成和修复这种NP特异性细胞外基质的能力。我们以前已经证明，分子组成和底物硬度的环境线索可以被操纵来促进NP细胞的提升基质合成。特别是，我们已经开发出一种层粘连蛋白呈现水凝胶，它可以促进健康的、生物合成活性的NP细胞的许多分子标记物(例如，短臂、整合素3、层粘连蛋白和升高的SGAG)的表达增加，但只有当交联体是“软的”(&lt；0.5kpa)而不是“硬的”(&gt；0.9kpa)时。我们的总体假设是，“柔软”的僵硬和层粘连蛋白参与的环境线索可以被用来促进健康的、生物合成活性的NP细胞表型的重新表达，用于成年、退行性人类NP的细胞。在具体目标1中，我们将确定年轻和/或成年变性的人NP细胞在不同硬度(0.3-50 kpa)的层粘连蛋白功能化聚乙烯(PEG)水凝胶上培养后，是否有NP特异性分子标记的表达增加，NP特异性基质生物合成增加，以及NP相关转录因子活性增加。NP特异性标记物将根据脊柱研究兴趣小组(Risbud等人)的一篇共识论文的指导，用mRNA、蛋白质和生化分析来测量。2015年，乔尔)。在特定的目标2中，我们将确定细胞识别多肽是否有能力促进年轻和/或成年变性的人NP细胞在多肽功能化的聚乙二醇水凝胶上的NP特异性标记的表达。我们已经鉴定了四种细胞识别多肽，它们来自整合素结合域和层粘连蛋白衍生结构域，当连接到“软”水凝胶底物上时，它们促进了人NP细胞的附着和SGAG合成的增加。我们将改变这四种多肽在特定目标1确定的最佳硬度的聚乙二醇底物上的比例，并随着时间的推移测量NP特异性分子标记的表达(对于特定目标1)。在具体目标3中，我们测试了最优的聚乙二醇层粘连蛋白或聚乙二醇多肽水凝胶是否能够保存健康的NP细胞表型的重新表达，以使退变的人NP细胞在运送到退变的椎间盘的病理环境中后重新表达。成年的变性NP细胞将在聚乙二醇层粘连蛋白或聚乙二醇肽底物上进行预处理，然后以可注射的原位交联型聚乙二醇水凝胶作为细胞载体，在裸鼠模型中将其送入退化的椎间盘中。将评估细胞停留时间(发光)、椎间盘高度和NP特异性标记物，以测试聚乙二醇脂微球或聚乙二醇多肽水凝胶是否能够支持这些退化的髓核细胞在原生盘中保存健康的NP细胞表型。这项研究的完成将识别能够促进健康的、生物合成活性的NP细胞重新表达的环境线索，并将其应用于原代成体NP细胞和祖细胞，以用于细胞介导的退变椎间盘的再生。

 DESCRIPTION (provided by applicant): Nucleus pulposus (NP) cells of the intervertebral disc are derived from notochord and synthesize a soft, gelatinous matrix containing numerous collagen, proteoglycan and laminin species. With age, NP cells become more fibroblast-like and lose their ability to synthesize and repair this NP-specific extracellular matrix. We have previously shown that environmental cues of molecular composition and substrate stiffness can be manipulated to promote elevated matrix synthesis for the NP cell. In particular, we have developed a laminin-presenting hydrogel that can promote increased expression for many molecular markers of the healthy, biosynthetically active NP cell (e.g, brachyury, integrin 3, laminin, and elevated sGAG), but only when crosslinked to be "soft" (<0.5 kPa) as opposed to "stiff" (>0.9 kPa). Our overall hypothesis is that environmental cues of "soft" stiffness and laminin engagement can be exploited to promote re-expression of the healthy, biosynthetically active NP cell phenotype for cells of the adult, degenerate human NP. In Specific Aim 1, we will determine if young and/or adult degenerate human NP cells have elevated expression of NP-specific molecular markers, elevated NP-specific matrix biosynthesis, and NP-related transcription factor activity following culture upon laminin-functionalized polyethylene (PEG) hydrogels of varying stiffness (0.3 -50 kPa). NP-specific markers will be measured with mRNA, protein and biochemical assays following guidance from a consensus paper by the Spine Research Interest Group (Risbud et al. 2015, JOR). In Specific Aim 2, we will determine if cell recognition peptides have an ability to promote elevated expression of NP-specific markers for young and/or adult degenerate human NP cells when cultured upon peptide-functionalized PEG hydrogels. We have identified four cell recognition peptides derived from integrin-binding and laminin-derived domains that promote human NP cell attachment and elevated sGAG synthesis when attached to "soft" hydrogel substrates. We will vary ratios of these four peptides upon PEG substrates of optimal stiffness identified from Specific Aim 1, and measure the expression of NP-specific molecular markers over time (as for Specific Aim 1). In Specific Aim 3, we test if an optimal PEG-laminin or PEG-peptide hydrogel can preserve the re-expression of the healthy NP cell phenotype for degenerate human NP cells following delivery to the pathological environment of the degenerated intervertebral disc. Adult, degenerate NP cells will be "preconditioned" upon PEG-laminin or PEG- peptide substrates, then delivered into degenerated discs in a nude rat model with an injectable, in situ crosslinking version of the PEG hydrogel as a cell carrier. Cell residence time (luminescence), disc height, and NP-specific markers will be evaluated to test if the PEG-LM or PEG-peptide hydrogel can support preservation of the healthy NP cell phenotype for these degenerate NP cells in the native disc. Completion of this study would identify environmental cues that can promote re-expression of a healthy, biosynthetically active NP cell with application to primary adult NP cells and progenitor cells for cell-mediated regeneration of the degenerate disc.