Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis

Yoojin Seo, Youngmee Jung, Soo Hyun Kim

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO2-EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO2 reactor and decellularized at 37 °C and 350 bar. After scCO2-EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO2-EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n = 6 in each group: collagen, scCO2-EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for angiogenesis analysis. Consequently, blood vessel formation and density of vWF and α-SMA in the scCO2-EtOH group were significantly greater than that in the collagen group. Here we suggest that heart-derived decellularized extracellular matrix (dECM) with scCO2-EtOH treatment is a highly promising angiogenic material for healing in ischemic disease. Statement of Significance: Supercritical carbon dioxide (scCO2) in a supercritical phase has low viscosity and high diffusivity between gas and liquid properties and is known to be affordable, non-toxic, and eco-friendly. Therefore, scCO2 extraction technology has been extensively used in commercial and industrial fields. Recently, decellularized extracellular matrix (dECM) was applied to tissue engineering and regenerative medicine as a scaffold, therapeutic material, and bio-ink for 3D printing. Moreover, the general decellularization method using detergents has limitations including eliminating tissue-derived ECM components and disrupting their structures after decellularization. To overcome these limitations, heart tissues were treated with scCO2-EtOH for decellularization, resulting in preserving of tissue due to the various ECM and angiogenic factors derived. In addition, initiation of angiogenesis was highly induced even after 3 days of injection.

Original languageEnglish
JournalActa Biomaterialia
DOIs
Publication statusAccepted/In press - 2017 Jan 1
Externally publishedYes

Fingerprint

Military electronic countermeasures
Hydrogel
Hydrogels
Carbon Dioxide
Carbon dioxide
Extracellular Matrix
Detergents
Tissue
Collagen
Angiogenesis Inducing Agents
Tissue Engineering
Scaffolds (biology)
Fluorescent Antibody Technique
Tissue engineering
Angiogenic Proteins
Rats
Staining and Labeling
Ink
Regenerative Medicine
Proteins

Keywords

  • Angiogenesis
  • Decellularization
  • ECM hydrogel
  • Heart decellularized ECM (dECM)
  • Supercritical carbon dioxide

ASJC Scopus subject areas

  • Biotechnology
  • Biomaterials
  • Biochemistry
  • Biomedical Engineering
  • Molecular Biology

Cite this

Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis. / Seo, Yoojin; Jung, Youngmee; Kim, Soo Hyun.

In: Acta Biomaterialia, 01.01.2017.

Research output: Contribution to journalArticle

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N2 - Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO2-EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO2 reactor and decellularized at 37 °C and 350 bar. After scCO2-EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO2-EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n = 6 in each group: collagen, scCO2-EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for angiogenesis analysis. Consequently, blood vessel formation and density of vWF and α-SMA in the scCO2-EtOH group were significantly greater than that in the collagen group. Here we suggest that heart-derived decellularized extracellular matrix (dECM) with scCO2-EtOH treatment is a highly promising angiogenic material for healing in ischemic disease. Statement of Significance: Supercritical carbon dioxide (scCO2) in a supercritical phase has low viscosity and high diffusivity between gas and liquid properties and is known to be affordable, non-toxic, and eco-friendly. Therefore, scCO2 extraction technology has been extensively used in commercial and industrial fields. Recently, decellularized extracellular matrix (dECM) was applied to tissue engineering and regenerative medicine as a scaffold, therapeutic material, and bio-ink for 3D printing. Moreover, the general decellularization method using detergents has limitations including eliminating tissue-derived ECM components and disrupting their structures after decellularization. To overcome these limitations, heart tissues were treated with scCO2-EtOH for decellularization, resulting in preserving of tissue due to the various ECM and angiogenic factors derived. In addition, initiation of angiogenesis was highly induced even after 3 days of injection.

AB - Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO2-EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO2 reactor and decellularized at 37 °C and 350 bar. After scCO2-EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO2-EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n = 6 in each group: collagen, scCO2-EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for angiogenesis analysis. Consequently, blood vessel formation and density of vWF and α-SMA in the scCO2-EtOH group were significantly greater than that in the collagen group. Here we suggest that heart-derived decellularized extracellular matrix (dECM) with scCO2-EtOH treatment is a highly promising angiogenic material for healing in ischemic disease. Statement of Significance: Supercritical carbon dioxide (scCO2) in a supercritical phase has low viscosity and high diffusivity between gas and liquid properties and is known to be affordable, non-toxic, and eco-friendly. Therefore, scCO2 extraction technology has been extensively used in commercial and industrial fields. Recently, decellularized extracellular matrix (dECM) was applied to tissue engineering and regenerative medicine as a scaffold, therapeutic material, and bio-ink for 3D printing. Moreover, the general decellularization method using detergents has limitations including eliminating tissue-derived ECM components and disrupting their structures after decellularization. To overcome these limitations, heart tissues were treated with scCO2-EtOH for decellularization, resulting in preserving of tissue due to the various ECM and angiogenic factors derived. In addition, initiation of angiogenesis was highly induced even after 3 days of injection.

KW - Angiogenesis

KW - Decellularization

KW - ECM hydrogel

KW - Heart decellularized ECM (dECM)

KW - Supercritical carbon dioxide

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