Engineering Microenvironments for the Nucleus Pulposus Cell

 描述（由申请人提供）：椎间盘的髓核（NP）细胞来源于脊索，并合成含有大量胶原蛋白、蛋白聚糖和层粘连蛋白的柔软凝胶状基质。随着年龄的增长，NP细胞变得更像成纤维细胞，失去了合成和修复这种NP特异性细胞外基质的能力。我们以前已经表明，环境线索的分子组成和基板刚度可以操纵，以促进提高NP细胞的基质合成。特别地，我们已经开发了层粘连蛋白呈递水凝胶，其可以促进健康的生物合成活性NP细胞的许多分子标志物（例如，短尾畸形、整联蛋白β 3、层粘连蛋白和升高的sGAG）的表达增加，但仅当交联为“软”（<0.5kPa）而不是“硬”（> 0.9kPa）时。我们的总体假设是，可以利用“软”刚度和层粘连蛋白接合的环境线索来促进成年退化的人NP的细胞的健康的、生物合成活性的NP细胞表型的再表达。在具体目标1中，我们将确定在不同刚度（0.3 - 50 kPa）的层粘连蛋白官能化聚乙烯（PEG）水凝胶上培养后，年轻和/或成年退化人NP细胞是否具有NP特异性分子标记物的表达升高、NP特异性基质生物合成升高和NP相关转录因子活性。根据脊柱研究兴趣小组（Risbud et al. 2015，JOR）共识论文的指导，将采用mRNA、蛋白质和生化测定法测量NP特异性标志物。在具体目标2中，我们将确定当在肽官能化的PEG水凝胶上培养时，细胞识别肽是否具有促进年轻和/或成年简并人NP细胞的NP特异性标志物的表达升高的能力。我们已经鉴定了四种来源于整合素结合和层粘连蛋白衍生结构域的细胞识别肽，其在附着于“软”水凝胶基质时促进人NP细胞附着和升高的sGAG合成。我们将改变这四种肽在从特异性目标1鉴定的最佳刚度的PEG底物上的比例，并测量NP特异性分子标记物随时间的表达（如特异性目标1）。在具体目标3中，我们测试了最佳的PEG-层粘连蛋白或PEG-肽水凝胶是否可以在递送到变性椎间盘的病理环境后保留变性人NP细胞的健康NP细胞表型的再表达。成年的退化NP细胞将在PEG-层粘连蛋白或PEG-肽底物上进行“预处理”，然后用可注射的原位交联形式的PEG水凝胶作为细胞载体递送到裸鼠模型中的退化椎间盘中。将评价细胞停留时间（发光）、椎间盘高度和NP特异性标志物，以测试PEG-LM或PEG-肽水凝胶是否可以支持天然椎间盘中这些退化NP细胞的健康NP细胞表型的保存。这项研究的完成将确定环境的线索，可以促进一个健康的，生物合成活性的NP细胞与应用程序的主要成人NP细胞和祖细胞的细胞介导的再生的退化光盘的重新表达。

 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.