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（由适用提供）：椎间盘的细胞核（NP）细胞源自脊索，并合成一个含有许多胶原蛋白，蛋白聚糖和层粘蛋白的柔软的胶状基质。随着年龄的增长，NP细胞变得更像成纤维细胞，并失去了合成和修复该NP特异性细胞外基质的能力。我们先前已经表明，可以操纵分子组成和底物刚度的环境提示，以促进NP细胞的升高基质合成。 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).我们的总体假设是，可以探索“软”刚度和层粘连蛋白参与度的环境线索，以促进成人成人细胞的健康，生物合成活性的NP细胞表型的重新表达。在特定目标1中，我们将确定年轻和/或成人退化的人NP细胞在NP特异性分子标记物的表达是否升高，NP特异性基质生物合成升高以及在层粘连蛋白官能化的聚乙烯（PEG）水凝胶上培养后培养后NP相关的转录因子活性是否升高。在脊柱研究兴趣组（Risbud etal。2015，JOR）指导的指导下，将用mRNA，蛋白质和生化测定法测量NP特异性标记。在特定的目标2中，我们将确定细胞识别petides是否具有促进年轻和/或成人退化的NP特异性标志物在肽官能化的PEG水凝胶上培养时的能力。我们已经确定了来自整联蛋白结合和层粘连蛋白衍生的结构域衍生的四个细胞识别肽，这些肽促进了人类NP细胞的附着和升高的SGAG合成时，当附着在“软”水凝胶底物上时。我们将在从特定目标1中鉴定出的最佳刚度的PEG底物上改变这四个肽的比例，并随着时间的推移测量NP特异性分子标记的表达（与特定目标1一样）。在特定的目标3中，我们测试了最佳的PEG-氯胺酮或PEG肽水凝胶是否可以保留健康的NP细胞表型的重新表达，以便在递送到退化的椎间盘椎间盘病理环境后退化的人NP细胞。成年的，退化的NP细胞将在PEG层蛋白或PEG肽底物上“预处理”，然后在裸大鼠模型中递送到具有可注射的原位交联版本的PEG水凝胶作为细胞载体的裸大鼠模型中。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天然盘中这些退化NP细胞的健康NP细胞表型保存。这项研究的完成将确定可以促进健康，生物合成活性NP细胞的环境线索，并应用于原代成年NP细胞和祖细胞中，以用于细胞介导的变性椎间盘的再生。