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Cited by 470Hydrogels: synthesis, properties, applicationsCellular Mechanics and InteractionsTissue Engineering and Regenerative Medicine

Student award winner in the undergraduate category for the society of biomaterials 9th World Biomaterials Congress, Chengdu, China, June 1–5, 2012: The effects of substrate stiffness on the in vitro activation of macrophages and in vivo host response to poly(ethylene glycol)‐based hydrogels

Open access
Blakney, Anna K., Swartzlander, Mark D., Bryant, Stephanie J.·2012·Journal of Biomedical Materials Research Part A
DOI 10.1002/jbm.a.34104PMC PMC3339197
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Abstract

Poly(ethylene glycol) (PEG) hydrogels, modified with RGD, are promising platforms for cell encapsulation and tissue engineering. While these hydrogels offer tunable mechanical properties, the extent of the host response may limit their in vivo applicability. The overall objective was to characterize the effects of hydrogel stiffness on the in vitro macrophage response and in vivo host response. We hypothesized that stiffer substrates induce better attachment, adhesion, and increased cell spreading, which elevates the macrophage classically activated phenotype and leads to a more severe foreign body reaction (FBR). PEG‐RGD hydrogels were fabricated with compressive moduli of 130, 240, and 840 kPa, and the same RGD concentration. Hydrogel stiffness did not impact macrophage attachment, but elicited differences in cell morphology. Cells retained a round morphology on 130 kPa substrates, with localized and dense F‐actin and localized α V integrin stainings. Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F‐actin, and greater α V integrin staining. When stimulated with lipopolysaccharide, macrophages had a classical activation phenotype, with increased expression of TNF‐α, IL‐1β, and IL‐6, however the degree of activation was significantly reduced with the softest hydrogels. A FBR ensued in response to all hydrogels when implanted subcutaneously in mice, but 28 days postimplantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels. In conclusion, hydrogels with lower stiffness led to reduced macrophage activation and a less severe and more typical FBR, and therefore are more suited for in vivo tissue engineering applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

AI snapshot

Written from the paper’s full text.

TL;DR

This study tested whether the stiffness of RGD-modified PEG hydrogels changes macrophage behavior in culture and the foreign body reaction after subcutaneous implantation in mice. Softer 130 kPa hydrogels reduced classical macrophage activation and produced a less severe, more typical foreign body reaction than stiffer 240 and 840 kPa hydrogels.

Key findings

  • Hydrogel stiffness did not affect macrophage attachment, as DNA content was similar across gels, but it changed morphology: cells on 130 kPa remained round with dense, localized F-actin and αV integrin staining, whereas cells on 240 and 840 kPa spread more, formed filopodia, and showed greater αV staining.
  • After LPS stimulation, pro-inflammatory genes TNF-α, IL-1β, and IL-6 were elevated, and expression was generally highest on the stiffest gels. At 24 hours, 840 kPa gels had 2-fold higher IL-1β (p<0.0001) and 4.5-fold higher IL-6 (p=0.0001) than 130 kPa gels, and 1.4-fold higher IL-1β (p=0.0364) and 6.5-fold higher IL-6 (p<0.0001) than 240 kPa gels; IL-10 was 2.6-fold higher on 840 kPa than 130 kPa (p=0.0004), while Arginase I was 150-fold higher on 130 kPa than 840 kPa at 24 hours (p=0.0474).
  • At 28 days after implantation, the macrophage layer at the implant surface was thinnest for 130 kPa gels: about 30 μm dorsally and 35 μm ventrally, versus about 74 μm and 139 μm for 240 kPa gels and about 88 μm and 208 μm for 840 kPa gels. The 240 and 840 kPa gels had significantly thicker macrophage layers than 130 kPa gels (p<0.05), though 840 kPa was not significantly different from 240 kPa; fibrous capsules formed around all gels, with a trend toward thicker capsules on stiffer gels.

Methods

  • PEG-RGD hydrogels were photopolymerized from 10%, 20%, or 40% PEG-dA with the same 2.5 mM monoacrylated PEG-YRGDS concentration, yielding compressive moduli of 130, 240, and 840 kPa.
  • In vitro, RAW 264.7 macrophages were used for attachment and morphology studies, and primary bone marrow-derived murine macrophages were used for qRT-PCR analysis of phenotype with or without 1 μg/ml LPS at 4, 8, or 24 hours; attachment was measured by PicoGreen dsDNA, and F-actin/αV integrin were imaged by confocal microscopy.
  • In vivo, hydrogels were implanted subcutaneously in 6–8-week-old male C57BL/6 mice, excised at 28 days, and analyzed by Masson’s trichrome and Mac3 immunohistochemistry; inflammatory cell layer thickness was measured semi-quantitatively with ImageJ, and data were analyzed by ANOVA with Tukey post-hoc tests.

Limitations

  • The paper states that the exact mechanisms linking substrate stiffness to macrophage activation remain unclear, that Arginase I results do not allow definitive conclusions, that fibrous capsule boundaries were not always defined enough for quantification, and that hydrogel swelling is inversely related to stiffness and could also influence the in vivo response, though the authors argue this is unlikely.
  • Evident from the text: the in vitro activation work relies on LPS stimulation and two different macrophage sources, the in vivo study uses only a 28-day endpoint, and the histological host response is assessed semi-quantitatively in a mouse model, so earlier reaction dynamics and full clinical translation are not captured.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1002/jbm.a.34104

Cites

62 of 62 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 67 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The degree of macrophage activation after LPS stimulation was significantly reduced on the softest hydrogels.

    “however the degree of activation was significantly reduced with the softest hydrogels.”
    Quote in the paperSubstantial claim
  • Findingown data

    At 28 days after implantation, the macrophage layer at the implant surface was significantly lower for the softest hydrogels.

    “28 days post-implantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels.”
    Quote in the paperSubstantial claim
  • Findingown data

    Hydrogel stiffness did not affect macrophage attachment.

    “Hydrogel stiffness did not impact macrophage attachment”
    • Also stated in: § In vitro macrophage attachment and morphology · plain
      “macrophage density was similar among all three hydrogel substrates, as measured by total DNA content.”
    Quote in the paperSubstantial claim
  • Findingown data

    Hydrogel stiffness produced differences in macrophage morphology.

    “but elicited differences in cell morphology”
    • Also stated in: § In vitro macrophage attachment and morphology · plain
      “The F-actin organization also exhibited visual differences as a function of substrate stiffness.”
    Quote in the paperSubstantial claim
  • Findingown data

    At 24 hours with LPS, Arginase I expression was 150-fold higher on 130 kPa gels than 840 kPa gels (p=0.0474).

    “At 24 hours, the 130 kPa gel had 150-fold higher (p=0.0474) expression over the 840 kPa gels.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 41
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 21

  • May combine two claims.

    Findingown data

    Without inflammatory co-stimulants, mean relative expression of TNF-α, IL-1β, and IL-6 decreased with increasing hydrogel stiffness, but the decrease was significant only for IL-1β between 130 and 840 kPa gels (p=0.0356).

    “For all genes, mean relative expression values decreased with increasing hydrogel stiffness but this decreases was only significant for IL-1β and between 130 and 840 kPa gels (p=.0356).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On stiffer substrates, macrophages were more spread, with filopodia, more defined F-actin, and greater αV integrin staining than on 130 kPa substrates.

    “Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F-actin, and greater αV integrin staining.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The equilibrium swelling ratio decreased with increasing PEG-dA concentration, with values ranging from 4.6 to 10.

    “The equilibrium swelling ratio decreased with increasing PEG-dA concentration with values thatranged from 4.6 to 10.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Arginase I mean relative expression was highest for the 240 kPa gels, but not significantly.

    “Mean relative expression values for Arginase I expression were highest for the 240 kPa gels, but not significantly.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    With LPS stimulation, culture time and hydrogel stiffness were not significant factors for Arginase I expression.

    “For Arginase I expression, culture time and hydrogel stiffness were not significant factors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The tangent modulus under compression increased with increasing PEG-dA concentration, with values of 130, 240, and 840 kPa.

    “The tangent modulus under compression increased with increasing PEG-dA concentration with values of 130, 240 and 840 kPa.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    With LPS stimulation, culture time and hydrogel stiffness were both factors affecting IL-10 expression.

    “For IL-10 expression, culture time (p<0.0001) and hydrogel stiffness (p<0.0001) were both factors affecting its expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 24 hours with LPS, IL-6 expression was 4.5-fold higher on 840 kPa gels than 130 kPa gels (p=0.0001) and 6.5-fold higher than 240 kPa gels (p<0.0001).

    “at 24 hours IL-1β and IL-6 expressions for the 840 kPa gels were 2-fold (p<0.0001) and 4.5-fold (p=0.0001) higher than the 130 kPa gels, respectively and 1.4-fold (p=0.0364) and 6.5-fold (p<0.0001) higher than the 240 kPa gels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 24 hours with LPS, IL-1β expression was 2-fold higher on 840 kPa gels than 130 kPa gels (p<0.0001) and 1.4-fold higher than 240 kPa gels (p=0.0364).

    “at 24 hours IL-1β and IL-6 expressions for the 840 kPa gels were 2-fold (p<0.0001) and 4.5-fold (p=0.0001) higher than the 130 kPa gels, respectively and 1.4-fold (p=0.0364) and 6.5-fold (p<0.0001) higher than the 240 kPa gels.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    On 130 kPa substrates, macrophages remained round with dense F-actin and αV integrin staining localized near the nucleus.

    “Cells retained a round morphology on 130kPa substrates, with localized and dense F-actin and localized αV integrin stainings.”
    • Also stated in: § In vitro macrophage attachment and morphology · plain
      “For the 130 kPa substrate, cells were rounded, compact and the actin was centralized around the nucleus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The FBR for the softest hydrogels appeared largely stabilized by 28 days with a thin dense avascular fibrous capsule ~30-40 μm thick.

    “Furthermore, the FBR appeared to be largely stabilized by 28 days with a thin dense avascular fibrous capsule that was ~30–40 μm thick.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 28 days, 840 kPa gels had the thickest macrophage layer, about 88 μm on the dorsal side and 208 μm on the ventral side, significantly thicker than 130 kPa gels (p<0.05) but not 240 kPa gels.

    “The 840 kPa gels had the thickest layer of activated macrophages ~88 μm on the dorsal side and ~208 μm deep on the ventral side, which were significantly thicker than 130 kPa gels (p<0.05), but not the 240 kPa gels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The response was improved with 240 kPa hydrogels and results were similar to previous reports, but the FBR was still largely atypical.

    “The response was improved with 240 kPa and results were similar to that of our previous reports, but the FBR was still largely atypical.15”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] Lynn AD Blakney AK Kyriakides TR Bryant SJ Temporal progression of the host response to implanted poly(ethylene glycol)-based hydrogels Journal of Biomedical Materials Research Part A 2011 96A 4 621 631 21268236 PMC3091279 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    IL-10 expression was elevated, to a greater degree with increasing gel stiffness, but its peak appeared delayed compared to pro-inflammatory cytokines.

    “IL-10 expression was also elevated, and to a greater degree with increasing gel stiffness, but its peak appears to be delayed when compared to the pro-inflammatory cytokines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The expression of all three inflammatory cytokines was significantly impacted by gel stiffness and was lowest for the 130 kPa gel.

    “The level of expression of all three inflammatory cytokines was significantly impacted by gel stiffness and was lowest for the 130 kPa”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Arginase expression was not significantly affected during the experiment or by gel stiffness, but showed significantly higher levels at 24 hours for the 130 kPa over the 840 kPa gel.

    “Arginase expression, while not significantly affected during the course of the experiment or by gel stiffness did show significantly higher levels at 24 hours for the 130 kPa over the 840 kPa gel.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Arginase type I is up-regulated during wound healing by macrophages and is involved in collagen precursor synthesis.

  • May combine two claims.

    Findingown data

    During initial culture, hydrogel stiffness had minimal effects on macrophage phenotype, except IL-1β expression was significantly affected by gel stiffness and was lower with increasing substrate stiffness.

    “During the initial culture period, hydrogel stiffness had minimal affects on macrophage phenotype, with one notable exception where IL-1β expression was significantly affected by gel stiffness, and interestingly was lower in gels with increasing substrate stiffness.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Greater integrin staining was observed on stiffer substrates (240 and 840 kPa) compared to the softer substrate, correlating with increased cell spreading on stiffer substrates.

    “Most notably, greater staining was observed in the stiffer substrates (240 and 840 kPa) when compared to the softer substrate, a finding that correlated with the increased cell spreading observed on the stiffer substrates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In this study, F-actin on the stiffer substrates did not appear more organized, although it was not characteristic of stress fibers.

    “In this study, the F-actin on the stiffer substrates did not have an appearance that was more organized, although not characteristic of stress fibers.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The authors' results indicated stark differences in cell morphology due to hydrogel stiffness, which led to differences in F-actin cytoskeletal structure and integrin localization.

    “Our results indicated stark differences in cell morphology as a result of hydrogel stiffness, which led to differences in the F-actin cyotskeletal structure and integrin localization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On the stiffer substrates (240 and 840 kPa), cells appeared more spread with filopodia protruding and a more defined F-actin.

    “Contrarily on the stiffer substrates (240 and 840 kPa), the cells appeared more spread with evidence of filopodia protruding from the cell and a more defined F-actin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Substrate stiffness has a significant impact on many cells and their fate, including cytoskeleton rearrangement, migration, stem cell differentiation, and muscle cell contractility.

    “The stiffness of the underlying substrate has been shown to have a significant impact on numerous cells and their fate, such as cellular cyotskeleton rearrangement,22 cell migration,23 stem cell differentation21, and muscle cell contractility.24,25”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [22] Ghosh K Pan Z Guan E Ge SR Liu YJ Nakamura T Ren XD Rafailovich M Clark RAF Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties Biomaterials 2007 28 4 671 679 17049594 PMC3625367 source

      Not checked in the free run.

    • cites [23] Lo CM Wang HB Dembo M Wang YL Cell movement is guided by the rigidity of the substrate Biophysical Journal 2000 79 1 144 152 10866943 PMC1300921 source

      Not checked in the free run.

    • cites [21] Engler AJ Sen S Sweeney HL Discher DE Matrix elasticity directs stem cell lineage specification Cell 2006 126 4 677 689 16923388 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    PEG-based hydrogels can be functionalized with biological moieties in a controlled and systematic manner to create environments specific to a tissue engineering application.

    “They present a ‘blank slate,’ which can be functionalized with biological moieties in a controlled and systematic manner creating environments that are specific for the tissue engineering application.1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Tibbitt MW Anseth KS Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture Biotechnology and Bioengineering 2009 103 4 655 663 19472329 PMC2997742 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A strong early inflammatory reaction with a large band of macrophages at the hydrogel surface and elevated IL-1β expression occurs within two days post-implantation and persists throughout four weeks with little stabilization.

    “A strong early inflammatory reaction, characterized by a large band of macrophages at the hydrogel surface, ensues within two days post-implantation along with elevated levels of interleukin-1β expression in the cells at and near the implant interface. This reaction persists throughout the four weeks, with little sign of stabilization.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] Lynn AD Blakney AK Kyriakides TR Bryant SJ Temporal progression of the host response to implanted poly(ethylene glycol)-based hydrogels Journal of Biomedical Materials Research Part A 2011 96A 4 621 631 21268236 PMC3091279 source

      Not checked in the free run.

    • cites [16] Lynn AD Kyriakides TR Bryant SJ Characterization of the in vitro macrophage response and in vivo host response to poly(ethylene glycol)-based hydrogels Journal of Biomedical Materials Research Part A 2010 93A 3 941 953 19708075 source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Macrophage cell spreading and cytoskeleton stress are higher on stiffer substrates.

    “Fereol et al.27 reported that macrophage cell spreading and cytoskeleton stress were higher on stiffer substrates.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] Fereol S Fodil R Labat B Galiacy S Laurent VM Louis B Isabey D Planus E Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties Cell Motility and the Cytoskeleton 2006 63 6 321 340 16634082 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Macrophages sense substrate stiffness, implying that stiffness may play a role in the foreign body reaction.

    “These reports provide evidence that macrophages indeed sense the stiffness of a substrate, implying that stiffness may play a role in the FBR.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [26] Beningo KA Lo CM Wang YL Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions Methods in Cell-Matrix Adhesion 2002 69 325 339 12071003

      Not checked in the free run.

    • cites [27] Fereol S Fodil R Labat B Galiacy S Laurent VM Louis B Isabey D Planus E Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties Cell Motility and the Cytoskeleton 2006 63 6 321 340 16634082 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    PEG hydrogels formed from PEG-diacrylate contain polyacrylate kinetic chains crosslinked by PEG, and these kinetic chains confer hydrophobicity and may cause nonspecific protein adsorption that elicits a foreign body reaction.

    “PEG hydrogels formed from PEG-diacrylate are comprised of polyacrylate kinetic chains that are crosslinked by PEG. The kinetic chains confer some degree of hydrophobicity and may lead to nonspecific protein adsorption, thus eliciting a FBR.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    To isolate the effects of substrate stiffness, hydrogels were formed with varying stiffness but the same RGD concentration.

    “To isolate the effects of substrate stiffness, hydrogels were formed with varying stiffness, but with same concentration of RGD.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Macrophages bind both stiff and soft particles to their membrane but only readily phagocytose stiff particles, leading to more frustrated phagocytes.

    “Beningo et al.26 reported that macrophages bind both stiff and soft particles to their cell membrane, but only readily phagocytose stiff particles, leading to more frustrated phagocytes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Beningo KA Lo CM Wang YL Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions Methods in Cell-Matrix Adhesion 2002 69 325 339 12071003

      Not checked in the free run.

  • May combine two claims.

    Premiseown data

    Incorporating the cell adhesion moiety RGD into PEG hydrogels led to significant improvements in reducing macrophage activation in vitro and the foreign body reaction in vivo, although a foreign body reaction still occurred.

    “The incorporation of a biologically active molecule into the PEG hydrogel, specifically the cell adhesion moiety RGD, led to significant improvements in the reduction of macrophage activation as seen in vitro and the FBR in vivo, although a FBR still ensued.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The severity of the foreign body reaction depends on numerous factors, including material chemistry and topography.

    “its severity depends on numerous factors, such as material chemistry18 and topography.19”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [18] Schutte RJ Parisi-Amon A Reichert WM Cytokine profiling using monocytes/macrophages cultured on common biomaterials with a range of surface chemistries Journal of Biomedical Materials Research Part A 2009 88A 1 128 139 18260130 PMC4070304 source

      Not checked in the free run.

    • cites [19] Chen SL Jones JA Xu YG Low HY Anderson JM Leong KW Characterization of topographical effects on macrophage behavior in a foreign body response model Biomaterials 2010 31 13 3479 3491 20138663 PMC2837101 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The foreign body reaction is thought to be a series of cell-material surface-mediated interactions beginning with non-specific protein adsorption followed by macrophage recruitment, attachment, and activation.

    “The FBR is thought to be a series of cell-material surface-mediated interactions. It begins with non-specific protein adsorption followed by macrophage recruitment, attachment to the biomaterial, and activation.10,11”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] Anderson JM Rodriguez A Chang DT Foreign body reaction to biomaterials Seminars in Immunology 2008 20 2 86 100 18162407 PMC2327202 source

      Not checked in the free run.

    • cites [11] Ratner BD Bryant SJ Biomaterials: Where we have been and where we are going Annual Review of Biomedical Engineering 2004 6 41 75 15255762 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Custom primers were validated for efficiency and stability of the house-keeping gene L32.

    “Custom primers were designed using Primer Express 3.0 software (Applied Biosystems) and validated for both efficiency and stability of the house-keeping gene L32, which encodes a ribosomal protein.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Others have reported reduced IL-1β production by human monocyte/macrophages with increased cell spreading when cultured without inflammatory stimulants.

    “Others have recently reported reduced IL-1β production by human monocyte/macrophages with increased cell spreading when cultured in the absence of an inflammatory stimulants.45”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Collie AMB Bota PCS Johns RE Maier RV Stayton PS Differential monocyte/macrophage interleukin-1 beta production due to biomaterial topography requires the beta 2 integrin signaling pathway Journal of Biomedical Materials Research Part A 2011 96A 1 162 169 21105164 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Others have reported that RGD-mediated binding events reduce inflammatory cytokine production.

    “Similarly, others have reported reduced inflammatory cytokine production by RGD-mediated binding events.38,39”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [38] Graves KL Roman J Fibronectin modulates expression of interleukin-1 beta and its receptor antagonist in human mononuclear cells American Journal of Physiology-Lung Cellular and Molecular Physiology 1996 271 1 L61 L69 8760133 source

      Not checked in the free run.

    • cites [39] Simms HH Damico R Bland KI Integrin stimulation regulates polymorphonuclear leukocytes inflammatory cytokine expression Annals of Surgery 1997 225 6 757 763 9230816 PMC1190884 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The authors previously reported that macrophages stimulated by LPS shift to a wound healing phenotype, but this shift was not apparent until 48 hours.

    “We have previously reported that macrophages after being stimulated by LPS shift to a wound healing phenotype but this shift was not apparent until 48 hours.50”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [50] Lynn AD Bryant SJ Phenotypic changes in bone marrow-derived murine macrophages cultured on PEG-based hydrogels activated or not by lipopolysaccharide Acta Biomaterialia 2011 7 1 123 132 20674808 PMC2967672 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    A fibrous capsule formed in all cases, but a defined boundary was not always observed, making quantification of the fibrous capsule layer difficult.

    “The formation of a fibrous capsule was apparent in all cases, although a defined boundary for the capsule was not always observed making it difficult to quantify the fibrous capsule layer.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    One PEG-RGD construct of each stiffness was placed into a single subcutaneous pocket in a random order.

    “One construct of PEG-RGD (10%, 20% or 40% (w/w) PEG-dA) was placed into a single pocket, in a random order.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The 130 kPa gel may shift macrophages towards a wound healing phenotype, perhaps more quickly than stiffer substrates.

    “While we cannot draw definitive conclusions, these results suggest that the 130 kPa may shift the macrophages towards a wound healing phenotype, and perhaps more quickly than the macrophages on stiffer substrates.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Arginase type I is up-regulated during wound healing by macrophages and is involved in collagen precursor synthesis.

  • May combine two claims.

    Interpretationown data

    The in vivo findings support the in vitro conclusions and the hypothesis that softer hydrogels lead to a reduced FBR, possibly by attenuating macrophage activation at the host-hydrogel interface.

    “Our in vivo findings further support our in vitro conclusions and support our hypothesis that softer hydrogels lead to a reduced FBR possible by attenuating macrophage activation at the host-hydrogel interface.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The integrin pairs α5β1, αvβ1, and αvβ3 are well known to bind RGD.

    “The integrin pairs, α5β1, αvβ1, and αvβ3, are well known integrin pairs that bind to RGD.41”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Ruoslahti E RGD and other recognition sequences for integrins Annual Review of Cell And Developmental Biology 1996 12 697 715 8970741 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The degree of hydrogel swelling, which is inversely related to mechanical properties through crosslinking density, may also play a role in the in vivo environment.

    “While we focus on hydrogel stiffness as the important external cue sensed by the macrophages, it is important to recognize the degree of hydrogel swelling, which is inversely related to the mechanical properties through crosslinking density, may also be playing a role particularly for the in vivo environment.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Hydrogel swelling is inversely related to mechanical properties through crosslinking density.

  • May combine two claims.

    Interpretationown data

    Hydrogel stiffness mediated cell adhesion and subsequently reduced or altered the number and/or spatial location of integrin-ligand bonds, despite similar RGD presentation.

    “This result suggests that hydrogel stiffness mediated cell adhesion and subsequently reduced or altered the number and/or spatial location of integrin-ligand bonds, despite the similar presentation of RGD ligands.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Many tissue engineering strategies using PEG hydrogels incorporate proteins or peptides to support cell adhesion and promote cell-material interactions.

    “Many tissue engineering strategies that use PEG hydrogels incorporate proteins or peptides to support cell adhesion and promote cell-material interactions.1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Tibbitt MW Anseth KS Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture Biotechnology and Bioengineering 2009 103 4 655 663 19472329 PMC2997742 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    RGD is the most widely studied cell adhesion peptide in tissue engineering.

    “RGD is the most widely studied cell adhesion peptide in tissue engineering34–36”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [34] Dvir T Timko BP Kohane DS Langer R Nanotechnological strategies for engineering complex tissues Nature Nanotechnology 2011 6 1 13 22 PMC4059057 21151110 source

      Not checked in the free run.

    • cites [35] Hubbell JA Biomaterials in tissue engineering Bio-Technology 1995 13 6 565 576 9634795 source

      Not checked in the free run.

    • cites [36] Sreejalekshmi KG Nair PD Biomimeticity in tissue engineering scaffolds through synthetic peptide modifications-Altering chemistry for enhanced biological response Journal of Biomedical Materials Research Part A 2011 96A 2 477 491 21171167 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In this study, macrophages were seeded onto PEG-RGD hydrogels in serum-free conditions to ensure direct interaction with RGD rather than secondary non-specific adsorbed protein.

    “In this study, macrophages were seeded onto the PEG-RGD hydrogels in serum-free conditions in an effort to ensure that macrophages interacted directly with RGD rather than any secondary non-specific adsorbed protein.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Serum-free culture conditions ensure that macrophages interact directly with RGD rather than with non-specifically adsorbed proteins.

  • May combine two claims.

    Premisecitation

    Previous findings by the authors showed that RGD attenuates, but does not completely eliminate, the foreign body response to PEG hydrogels.

    “Our previous findings showed that RGD is able to attenuate, although not completely, the FBR to PEG hydrogels.15,16”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] Lynn AD Blakney AK Kyriakides TR Bryant SJ Temporal progression of the host response to implanted poly(ethylene glycol)-based hydrogels Journal of Biomedical Materials Research Part A 2011 96A 4 621 631 21268236 PMC3091279 source

      Not checked in the free run.

    • cites [16] Lynn AD Kyriakides TR Bryant SJ Characterization of the in vitro macrophage response and in vivo host response to poly(ethylene glycol)-based hydrogels Journal of Biomedical Materials Research Part A 2010 93A 3 941 953 19708075 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Before implantation, all hydrogels were confirmed to be free of endotoxins.

    “Prior to implantation, all hydrogels were confirmed to be free of endotoxins.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In a simulated inflammatory environment, softer substrates help reduce the classically activated macrophage phenotype and shift to a wound healing phenotype.

    “Taken together, our in vitro results demonstrate that in a simulated inflammatory environment, softer substrates help to reduce the classically activated macrophage phenotype and shift to a wound healing phenotype.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cellular prestress development from these events is thought to trigger downstream events implicated in morphogenesis, physiology, and disease.

    “It is thought that the development of cellular prestress as a result of these events trigger a wide range of downstream events in the cell that have been implicated in morphogensis, physiology and disease.57”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [57] Hoffman BD Grashoff C Schwartz MA Dynamic molecular processes mediate cellular mechanotransduction Nature 475 7356 316 323 21776077 PMC6449687 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    When highly swollen hydrogels are implanted into a wound site, they may soak up proinflammatory cytokines present in the exudate at early wound stages, potentially leading to their prolonged presence and chronic inflammation.

    “When these highly swollen hydrogels are implanted into a wound site, they may ‘soak up’ many of the proinflammatory cytokines that are present in the exudate at the early stages of a wound and subsequently lead to their prolonged presence and chronic inflammation.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Hydrogels absorb many times their polymer weight in water and form loosely crosslinked networks that allow facile transport of water and other molecules.

  • May combine two claims.

    Interpretationreasoning

    Substrate stiffness, likely through integrin-mediated events, directs changes in macrophage morphology and cytoskeletal organization, which subsequently alters their reaction to PEG-RGD hydrogels.

    “Taken together, our findings suggest that substrate stiffness likely through integrin-mediated events direct changes in macrophage morphology and cytoskeletal organization, which subsequently alters their reaction to the PEG-RGD hydrogels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Macrophages appear more sensitive to stiffer substrates, while contractile adherent cells tend to be less sensitive when substrates are stiff.

    “Interestingly, macrophages appear to be more sensitive to stiffer substrates, while contractile adherent cells tend to be less sensitive when substrates are stiff.56”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Fereol S Fodil R Laurent VM Balland M Louis B Pelle G Henon S Planus E Isabey D Prestress and Adhesion Site Dynamics Control Cell Sensitivity to Extracellular Stiffness Biophysical Journal 2009 96 5 2009 2022 19254561 PMC2717368 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Macrophages, as part of their myeloid lineage, do not possess stress fibers, possibly because their interaction with surrounding matrix when not migrating is not a key part of their innate function.

    “It has been shown that macrophages, as part of their myeloid lineage, do not possess stress fibers,58 which may in large part be due to the fact that their interaction with their surrounding matrix, when not migrating, is not a key part of their innate function.29”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [58] Jones GE Allen WE Ridley AJ The Rho GTPases in macrophage motility and chemotaxis Cell Adhesion and Communication 1998 6 2–3 237 245 9823474 source

      Not checked in the free run.

    • cites [29] Mosser DM Edwards JP Exploring the full spectrum of macrophage activation Nature Reviews Immunology 2008 8 12 958 969 PMC2724991 19029990 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Overall, the authors' findings indicate that stiffness of the synthetic scaffold contributes to the severity of the FBR and is an external cue that should be considered when designing scaffolds for tissue engineering.

    “Overall, our findings indicate that stiffness of the synthetic scaffold will contribute to the severity of the FBR and is an external cue that should be considered when designing scaffolds for tissue engineering.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    For many cell types, cells sense substrate stiffness through integrin-mediated events involving focal adhesions, actin organization, and stress fiber development.

    “It has been shown, for many cell types, that cells sense the stiffness of their substrata through integrin-mediated events involving focal adhesions, actin organization and development of stress fibers.55–57”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [55] Wang N Tolic-Norrelykke IM Chen JX Mijailovich SM Butler JP Fredberg JJ Stamenovic D Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells American Journal of Physiology-Cell Physiology 2002 282 3 C606 C616 11832346 source

      Not checked in the free run.

    • cites [56] Fereol S Fodil R Laurent VM Balland M Louis B Pelle G Henon S Planus E Isabey D Prestress and Adhesion Site Dynamics Control Cell Sensitivity to Extracellular Stiffness Biophysical Journal 2009 96 5 2009 2022 19254561 PMC2717368 source

      Not checked in the free run.

    • cites [57] Hoffman BD Grashoff C Schwartz MA Dynamic molecular processes mediate cellular mechanotransduction Nature 475 7356 316 323 21776077 PMC6449687 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    For contractile adherent cells, F-actin develops into highly organized stress fibers as substrate stiffness increases, with this transition occurring on substrates as soft as ~10 kPa.

    “For contractile adherent cells, cells typically developed their F-actin into highly organized stress fibers as substrate stiffness increases but this transition occurs on substrates as soft as ~10 kPa.21”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Engler AJ Sen S Sweeney HL Discher DE Matrix elasticity directs stem cell lineage specification Cell 2006 126 4 677 689 16923388 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The stiffer the substrate, the stronger the focal adhesions and the more organized the stress fibers.

    “The overwhelming observations are that the stiffer the substrate, the stronger the focal adhesions are and the more organized the stress fibers are.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [55] Wang N Tolic-Norrelykke IM Chen JX Mijailovich SM Butler JP Fredberg JJ Stamenovic D Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells American Journal of Physiology-Cell Physiology 2002 282 3 C606 C616 11832346 source

      Not checked in the free run.

    • cites [56] Fereol S Fodil R Laurent VM Balland M Louis B Pelle G Henon S Planus E Isabey D Prestress and Adhesion Site Dynamics Control Cell Sensitivity to Extracellular Stiffness Biophysical Journal 2009 96 5 2009 2022 19254561 PMC2717368 source

      Not checked in the free run.

    • cites [57] Hoffman BD Grashoff C Schwartz MA Dynamic molecular processes mediate cellular mechanotransduction Nature 475 7356 316 323 21776077 PMC6449687 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The results support the hypothesis that stiffer PEG-RGD hydrogels lead to increased cell spreading with a more organized F-actin cytoskeleton network.

    “The results from this study support our hypothesis that stiffer PEG-RGD hydrogels lead to increased cell spreading with a more organized F-actin cyotskeleton network”
    Quote in the paperSubstantial claim
59 of 59

  • The paper relies on this without support.

    There is little prior knowledge about how substrate stiffness affects macrophages, their activation, and the foreign body reaction.

    “However, there is little known regarding how stiffness impacts macrophages and more specifically their activation and ultimately the FBR.”

  • The paper relies on this without support.

    The foreign body reaction occurs when nearly all non-biological materials are implanted.

    “We know that the FBR occurs when nearly all non-biological materials are implanted”

  • The paper relies on this without support.

    Arginase type I is up-regulated during wound healing by macrophages and is involved in collagen precursor synthesis.

    “The last gene investigated was Arginase type I, which is up-regulated during wound healing by macrophages and is involved in the synthesis of collagen precursors.”

  • The paper relies on this without support.

    The foreign body response will likely be a hurdle to regenerating tissues.

    “it will likely be a hurdle to regenerating tissues.”

  • The paper relies on this without support.

    Hydrogel swelling is inversely related to mechanical properties through crosslinking density.

    “the degree of hydrogel swelling, which is inversely related to the mechanical properties through crosslinking density”

  • The paper relies on this without support.

    Hydrogels absorb many times their polymer weight in water and form loosely crosslinked networks that allow facile transport of water and other molecules.

    “Because hydrogels absorb many times their polymer weight in water, they form loosely crosslinked networks that allow for facile transport of water and other molecules through the gels.”

  • The paper relies on this without support.

    Serum-free culture conditions ensure that macrophages interact directly with RGD rather than with non-specifically adsorbed proteins.

    “In this study, macrophages were seeded onto the PEG-RGD hydrogels in serum-free conditions in an effort to ensure that macrophages interacted directly with RGD rather than any secondary non-specific adsorbed protein.”

  • The paper relies on this without support.

    In vitro experiments provide a more controlled environment for studying macrophages than in vivo experiments.

    “To probe the effects of hydrogel stiffness on macrophage activation, we turned to in vitro experiments because they provided a more controlled environment for studying macrophages.”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • LPS-stimulated macrophages display an increase in inflammatory cytokine production with matrix stiffening (130, 240 and 840 kPa).

    “Blakney et al. found that LPS‐stimulated macrophages displayed an increase in inflammatory cytokine production with matrix stiffening (130, 240 and 840 kPa). 33”

    From Matrix stiffness regulates macrophage polarisation via the <scp>Piezo1‐YAP</scp> signalling axis (2024) · cites this paper as [33]

    The Results section reports that, in LPS-stimulated murine macrophages, inflammatory cytokine gene expression varied with PEG-RGD hydrogel stiffness and was generally higher on the 840 kPa gels than on the 130 kPa gels. The study measured mRNA expression, not cytokine protein production; it also does not establish a steady increase across all three stiffnesses. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Several studies have reported that stiffer substrates promote M1 polarisation.

    “Several studies have reported that stiffer substrates promote M1 polarisation.”

    From Soft tissue-mimicking hydrogel stiffness modulates polarisation of human monocyte-derived macrophages (2025) · cites this paper as [13]

    The Discussion reports that, under LPS stimulation, macrophages on the softest PEG-RGD hydrogel had lower expression of inflammatory cytokines characteristic of classical (M1) activation than those on stiffer gels. It also says hydrogel stiffness had minimal effects on macrophage phenotype during the initial culture period. The unqualified claim omits that inflammatory-stimulation condition. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • The stiffness-dependent trend observed in this study aligns with previous reports that increased stiffness promotes a shift towards M1-like macrophage phenotype.

    “The stiffness-dependent trend observed in our study aligns with several previous reports in which increased stiffness promoted a shift towards M1-like macrophage phenotype.”

    From Soft tissue-mimicking hydrogel stiffness modulates polarisation of human monocyte-derived macrophages (2025) · cites this paper as [13]

    The Discussion and Results report that, with LPS stimulation, murine macrophages on stiffer PEG-RGD hydrogels showed higher expression of cytokines associated with classical (M1-like) activation. The claim omits that inflammatory condition: without a stimulant, stiffness had little effect on phenotype, and IL-1β expression decreased as stiffness increased. Page numbers are unavailable in our copy. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Blakney et al. reported increased TNF-α, IL-1β, and IL-6 expression in macrophages cultured on stiff PEG gels (840 kPa).

    “Blakney et al. reported increased TNF-α, IL-1β, and IL-6 expression in macrophages cultured on stiff PEG gels (840 kPa) 13”

    From Soft tissue-mimicking hydrogel stiffness modulates polarisation of human monocyte-derived macrophages (2025) · cites this paper as [13]

    The Results and Discussion report that, with LPS stimulation, expression of TNF-α, IL-1β, and IL-6 was affected by gel stiffness and was lowest on the 130 kPa gels. The claim omits that inflammatory-stimulation condition: without it, the Results report that mean expression of all three decreased as stiffness increased. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

  • In both studies, softer gels (> 11 kPa) promoted an anti-inflammatory phenotype.

    “In both studies, softer gels (> 11 kPa) promoted an anti-inflammatory phenotype.”

    From Soft tissue-mimicking hydrogel stiffness modulates polarisation of human monocyte-derived macrophages (2025) · cites this paper as [13]

    The Discussion reports that softer gels reduced classical macrophage activation in vitro and suggests that the 130 kPa gel may shift macrophages toward a wound-healing phenotype. The in vivo Results report fewer activated macrophages around the 130 kPa gel than around stiffer gels, but do not establish an anti-inflammatory phenotype. The paper studies 130, 240, and 840 kPa gels and does not identify 11 kPa as a threshold. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Morphology assessment is dated at both 24 and 48 hours

    Minor

    The methods say the cells used for staining were fixed after 24 hours, whereas the results describe the morphology observations as occurring after 48 hours.

  • Macrophage-layer comparison reverses the reported measurements

    Minor

    The stated direction is opposite to the measurements: for every gel stiffness, the ventral macrophage layer is thicker than the dorsal layer.

  • Macrophage cell-line identifier changes in the results

    Minor

    The methods identify the attachment and morphology cell line as RAW 264.7, but the results call it RAW 246.7.

  • F-actin organization is described in opposite terms

    Minor

    The discussion says F-actin on stiffer substrates did not appear more organized, while the conclusion says stiffer hydrogels produced a more organized F-actin network.

4 of 4

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Anderson JM, 1983, Biocompatibility and the inflammatory response, Abstr Pap Am Chem Soc, 185, 99‐POLY
  2. Erickson CAR, 1989, Clinical Immunology

Cited by

Sorted by citations

Authors
Blakney, A. K., Swartzlander, M. D., & Bryant, S. J.
Affiliations
University of Colorado Boulder (US)
Corresponding author
Bryant, Stephanie J.
ORCID
Venue
Journal of Biomedical Materials Research Part A
Publisher
Wiley
Published
2012-03-07
Volume
100A
Issue
6
Pages
1375-1386
ISSN
  • 1549-3296 (ISSN-L)
  • 1552-4965
DOI
10.1002/jbm.a.34104
PMC
PMC3339197
OpenAlex
W1489759013
Cited by
470
References
62
Citations by year
2026: 22 · 2025: 40 · 2024: 41 · 2023: 32 · 2022: 47 · 2021: 65 · 2020: 42 · 2019: 43 · 2018: 41 · 2017: 33 · 2016: 20 · 2015: 18 · 2014: 14 · 2013: 8 · 2012: 4
FWCI
6.50
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations
  • Journal of Biomedical Materials Research Part A (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institute of Dental and Craniofacial Research (K22DE016608, R03DE019505)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Medicine › Hydrogels: synthesis, properties, applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Keywords
poly(ethylene glycol) hydrogels, hydrogel stiffness, macrophage activation, foreign body reaction, host response, cell adhesion, cell spreading, ITGAV, F-actin, lipopolysaccharide, tissue engineering scaffolds, cell encapsulation
MeSH terms
Animals, Awards and Prizes, Biocompatible Materials, Cell Adhesion, Cell Line, Gene Expression Regulation, Lipopolysaccharides, Macrophages, Male, Mice, Inbred C57BL, Oligopeptides, Peptide Fragments, Polyethylene Glycols, Tumor Necrosis Factor-alpha, Interleukin-6, Hydrogels, Mice, Interleukin-1beta, Mechanical Phenomena

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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