Development of a library of laminin-mimetic peptide hydrogels for control of nucleus pulposus cell behaviors.
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ABSTRACT:
SUBMITTER: Speer JE
PROVIDER: S-EPMC8211742 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Purpose: Degeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by reduced cellularity and a shift towards a fibroblast-like state. We have recently demonstrated an ability to manipulate the phenotypic expression of adult degenerative NP cells by culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides. In the present study, we seek to understand the transcriptome changes elicited by NP cell interactions with the presented hydrogel system. Methods: mRNA profiles of NP from 3 human tissue samples were cultured for 4 days on either tissue culture polysyrene (TCPS) or the functionalized gel before RNA was isolated and sequenced, using Illumina NovaSeq. Unaligned reads were trimmed based on quality score (minimum quality level (Phred) = 20, minimum read length = 25) and aligned to the whole human genome (STAR 2.6.1d; hg19) using Partek Flow software. This software suite was also used to calculate the counts/normalized counts of genes and to perform gene specific analysis (GSA), principle component analysis (PCA), hierarchical clustering, and gene set enrichment. Differentially regulated genes were considered to be those with a fold change value (gel/TCPS) that was greater than or equal to 2 or less than or equal to negative 2 and a p-value of less than or equal to 0.05. Results: The data corroborate and expand on the previous findings by demonstrating that degenerative adult human NP cells cultured upon these gels have upregulations in some markers of NP and notochordal cells but also downregulations of some NP-specific markers and several fibroblastic markers. Furthermore, through gene set enrichment analysis we have shown that pathways related to cell differentiation and notochord morphogenesis were upregulated in the gel condition. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as up or downregulated following periods of culture upon the gel condition. Conclusions: The data from this RNA-sequencing demonstrate that culture on laminin-mimetic-peptide presenting gels can modulate cell phenotype and promotes upregulation of NP and notochordal markers.
2021-02-01 | GSE154044 | GEO
Project description:INTRODUCTION: Thermoreversible hydrogels have potential in spine research as they provide easy injectability and mild gelling mechanism (by physical cross-link). The purpose of this study was to assess the potential of thermoreversible hyaluronan-based hydrogels (HA-pNIPAM) (pNIPAM Mn = 10, 20, 35 × 10(3) g mol(-1)) as nucleus pulposus cells (NPC) carrier. MATERIALS AND METHODS: Cytocompatibility (WST-1 assay), viability (trypan blue), morphology (toluidine blue), sulphated glycosaminoglycan synthesis (DMMB assay) and gene expression profile (real-time PCR) of bovine NPC cultured in HA-pNIPAM were followed for 1 week and compared to alginate gel bead cultures. The injectability and cell survival in a whole disc organ culture model were assessed up to day 7. RESULTS: All HA, HA-pNIPAM and their degradation products were cytocompatible to NPC. HA-pNIPAM hydrogels with no volume change upon gelling maintained NPC viability and characteristic rounded morphology. Glycosaminoglycan synthesis was similar in HA-pNIPAM and alginate gels. Following NPC expansion, both gels induced re-differentiation toward the NPC phenotype. Significant differences between the two gels were found for COLI, COLII, HAS1, HAS2 and ADAMTS4 but not for MMPs and TIMPs. Higher expression of hyaluronan synthases (HAS1, HAS2) and lower expression of COLI and COLII mRNA were noted in cells cultured in HA-pNIPAM (pNIPAM = 20 × 10(3)g mol(-1)). NPC suspension in HA-pNIPAM was injectable through a 22-G needle without loss of cell viability. Ex vivo, NPC viability was maintained in HA-pNIPAM for 1 week. CONCLUSION: A HA-pNIPAM composition suitable for nucleus pulposus repair that provides an injectable carrier for NPC, maintains their phenotype and promotes extracellular matrix generation was identified.
| S-EPMC3535207 | biostudies-literature
Project description:Purpose of review:Intervertebral discs (IVD) are derived from embryonic notochord and sclerotome. The nucleus pulposus is derived from notochord while other connective tissues of the spine are derived from sclerotome. This manuscript will review the past 5 years of research into IVD development. Recent findings:Over the past several years, advances in understanding the step-wise process that govern development of the nucleus pulposus and the annulus fibrosus have been made. Generation of tissues from induced or embryonic stem cells into nucleus pulposus and paraxial mesoderm derived tissues has been accomplished in vitro using pathways identified in normal development. A balance between BMP and TGF-? signaling as well as transcription factors including Pax1/Pax9, Mkx and Nkx3.2 appear to be very important for cell fate decisions generating tissues of the IVD. Summary:Understanding how the IVD develops will provide the foundation for future repair, regeneration, and tissue engineering strategies for IVD disease.
| S-EPMC6261384 | biostudies-literature
Project description:Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.
| S-EPMC4416732 | biostudies-literature
Project description:Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.
| S-EPMC4474637 | biostudies-literature
Project description:The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.
| S-EPMC10706138 | biostudies-literature
Project description:Cells of the nucleus pulposus have been observed to undergo a shift from their notochordal-like juvenile phenotype to a more fibroblast-like state with age and maturation. It has been demonstrated that culture of degenerative adult human nucleus pulposus cells upon soft (<1 kPa) full length laminin-containing hydrogel substrates promotes increased levels of a panel of markers associated with the juvenile nucleus pulposus cell phenotype. In the current work, we observed an ability to use soft polymeric substrates functionalized with short laminin-mimetic peptide sequences to recapitulate the behaviors elicited by soft, full-length laminin containing materials. Furthermore, our work suggests an ability to mimic features of soft systems through control of peptide density upon stiffer substrates. Specifically, results suggest that stiffer polymer-peptide hydrogel substrates can be used to promote the expression of a more juvenile-like phenotype for cells of the nucleus pulposus by reducing adhesive ligand presentation. Here we show how polymer stiffness combined with adhesive ligand presentation can be controlled to be supportive of nucleus pulposus cell phenotype and biosynthesis.
| S-EPMC7608136 | biostudies-literature
Project description:Degradation of the nucleus pulposus (NP) is an early hallmark of intervertebral disc degeneration. The capacity for endogenous regeneration in the NP is limited due to the low cellularity and poor nutrient and vascular supply. Towards restoring the NP, a number of biomaterials have been explored for cell delivery. These materials must support the NP cell phenotype while promoting the elaboration of an NP-like extracellular matrix in the shortest possible time. Our previous work with chondrocytes and mesenchymal stem cells demonstrated that hydrogels based on hyaluronic acid (HA) are effective at promoting matrix production and the development of functional material properties. However, this material has not been evaluated in the context of NP cells. Therefore, to test this material for NP regeneration, bovine NP cells were encapsulated in 1%w/vol HA hydrogels at either a low seeding density (20×10(6)cellsml(-1)) or a high seeding density (60×10(6)cellsml(-1)), and constructs were cultured over an 8week period. These NP cell-laden HA hydrogels showed functional matrix accumulation, with increasing matrix content and mechanical properties with time in culture at both seeding densities. Furthermore, encapsulated cells showed NP-specific gene expression profiles that were significantly higher than expanded NP cells prior to encapsulation, suggesting a restoration of phenotype. Interestingly, these levels were higher at the lower seeding density compared to the higher seeding density. These findings support the use of HA-based hydrogels for NP tissue engineering and cellular therapies directed at restoration or replacement of the endogenous NP.
| S-EPMC4274233 | biostudies-literature
Project description:Mechanical properties of tissue scaffolds have major effects on the morphology and differentiation of cells. In contrast to two-dimensional substrates, local biochemical and mechanical properties of three-dimensional hydrogels are difficult to control due to the geometrical confinement. We designed synthetic 3D hydrogels featuring complexes of four-arm poly(ethylene glycol) (PEG) and collagen mimetic peptides (CMPs) that form hydrogels via physical cross-links mediated by thermally reversible triple helical assembly of CMPs. Here we present the fabrication of various PEG-CMP 3D hydrogels and their local mechanical properties determined by particle tracking microrheology. Results show that CMP mediated physical cross-links can be disrupted by altering the temperature of the gel or by adding free CMPs that compete for triple helix formation. This allowed modulation of both bulk and local stiffness as well as the creation of stiffness gradients within the PEG-CMP hydrogel, which demonstrates its potential as a novel scaffold for encoding physicochemical signals for tissue formation.
| S-EPMC3006224 | biostudies-literature
Project description:Engineered Peptide-Functionalized Hydrogels Modulate RNA Transcriptome of Human Nucleus Pulposus Cells In Vitro
| PRJNA644918 | ENA