Engineering Microenvironments for the Nucleus Pulposus Cell

髓核细胞的工程微环境

• 批准号: 9228325
• 负责人: Lori A. Setton
• 金额: $ 30.2万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9228325
• 关键词: Adolescent; Adult; Age; Aging; Alpha Cell; Anabolism; Behavior; Binding; Biochemical; Biochemistry; Biocompatible Materials; Biological Assay; Biological Preservation; Brachyury protein; Cell Communication; Cell Culture Techniques; Cell Nucleus; Cell-Matrix Junction; Cells; Cellular Morphology; Characteristics; Collagen; Consensus; Cues; Custom; Data; Dimensions; Engineering; Environment; Extracellular Matrix; Family suidae; Fibroblasts; Flow Cytometry; Height; Human; Hydrogels; Image; Immunohistochemistry; In Situ; In Vitro; Injectable; Integrin Binding; Integrins; Interest Group; Intervertebral disc structure; Label; Laminin; Length; Luciferases; Maintenance; Measures; Mediating; Messenger RNA; Metabolism; Modeling; Molecular; Natural regeneration; Nude Rats; Operative Surgical Procedures; Paper; Pathologic; Pathology; Patients; Peptides; Phenotype; Polyethylenes; Proteins; Proteoglycan; Reporter; Research; Roentgen Rays; Stem cells; Testing; Time; Tissues; Transgenes; Vertebral column; Work; cost; crosslink; design; improved; in vivo; interest; intervertebral disk degeneration; laminin A; luminescence; molecular marker; notochord; nucleus pulposus; phenotypic biomarker; preconditioning; protein aminoacid sequence; public health relevance; receptor; repaired; residence; scoliosis; transcription factor; wasting; young adult

 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.

 描述（由申请人提供）：椎间盘的髓核（NP）细胞源自脊索，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并失去其功能。合成和修复这种 NP 特异性细胞外基质的能力我们之前已经表明，可以操纵分子组成和基质硬度的环境线索来促进基质合成的提高。特别是，我们开发了一种层粘连蛋白呈递水凝胶，可以促进健康、具有生物合成活性的 NP 细胞的许多分子标记物的表达增加（例如 brachyury、整合素 α3、层粘连蛋白和升高的 sGAG）。当交联为“软”（<0.5 kPa）而不是“硬”（>0.9 kPa）时，我们的总体假设是环境因素。可以利用“软”硬度和层粘连蛋白接合来促进成人退化人类 NP 细胞的健康、生物合成活性 NP 细胞表型的重新表达。在具体目标 1 中，我们将确定年轻和/或成人是否退化。人 NP 细胞在层粘连蛋白功能化聚乙烯 (PEG) 上培养后，NP 特异性分子标记的表达升高、NP 特异性基质生物合成和 NP 相关转录因子活性升高不同硬度 (0.3 -50 kPa) 的水凝胶将按照脊柱研究兴趣小组 (Risbud et al. 2015, JOR) 的共识论文的指导，通过 mRNA、蛋白质和生化测定进行测量。 2，我们将确定当在肽功能化的 PEG 上培养时，细胞识别肽是否能够促进年轻和/或成年简并人类 NP 细胞的 NP 特异性标记物表达升高我们已经鉴定出四种来自整合素结合和层粘连蛋白衍生结构域的细胞识别肽，当连接到“软”水凝胶基底时，它们促进人 NP 细胞附着和 sGAG 合成。我们将改变这四种升高的肽在 PEG 基底上的比例。从特定目标 1 中确定最佳刚度，并测量 NP 特异性分子标记随时间的表达（对于特定目标 1，我们测试是否是最佳的）。 PEG-层粘连蛋白或PEG-肽水凝胶可以在递送至退化椎间盘的病理环境后保留退化人类NP细胞的健康NP细胞表型的重新表达，成年退化NP细胞将在PEG-的作用下“预处理”。层粘连蛋白或 PEG-肽底物，然后将 PEG 水凝胶的可注射原位交联版本作为细胞递送至裸鼠模型中的退变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，以测试 PEG-LM 或 PEG-肽水凝胶是否可以支持保存天然椎间盘中这些退化 NP 细胞的健康 NP 细胞表型。这项研究的完成将确定可促进健康、具有生物合成活性的 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，以实现退变椎间盘的细胞介导再生。