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1.
Biomaterials ; 278: 121156, 2021 11.
Article in English | MEDLINE | ID: mdl-34597900

ABSTRACT

Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated ß-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.


Subject(s)
Heat-Shock Proteins , Mesenchymal Stem Cells , Reactive Oxygen Species , Animals , Glucose , Hydrogels , Membrane Proteins , Osteogenesis , Palatine Tonsil , Proteomics , Rats
2.
J Biomed Mater Res A ; 109(10): 2001-2016, 2021 10.
Article in English | MEDLINE | ID: mdl-33818867

ABSTRACT

Various biomaterials have been used for bone and cartilage regeneration, and inflammation associated with biomaterial implantation is also increased. A 15-mer synthetic anti-inflammatory peptide (SAP15) was designed from human ß-defensin 3 to penetrate cells and induce intracellular downregulation of inflammation. The downregulation of inflammation was achieved by the binding of SAP15 to intracellular histone deacetylase (HDAC5). SAP15-mediated inhibition of inflammation was examined in vitro and in vivo using murine macrophages, human articular chondrocytes, and a collagen-induced arthritis (CIA) rat model. Surface plasmon resonance and immunoprecipitation assays indicated that SAP15 binds to HDAC5. SAP15 inhibited the lipopolysaccharide (LPS)-induced phosphorylation of intracellular HDAC5 and NF-κB p65 in murine macrophages. SAP15 treatment increased aggrecan and type II collagen expression and decreased osteocalcin expression in LPS-induced chondrocytes. Subcutaneous injection of SAP15-loaded sodium hyaluronic acid (HA) solution significantly decreased hind paw swelling, joint inflammation, and serum cytokine levels in CIA rats compared with the effects of sodium HA solution alone. The SAP15-loaded HA group exhibited preservation of cartilage and bone structure in CIA rat joints. Moreover, a more robust anti-inflammatory effect of the SAP15 loaded HA was observed than that of etanercept (an anti-tumor necrosis factor-alpha [TNF-α] antibody)-loaded HA. These findings suggest that SAP15 has an anti-inflammatory effect that is not controlled by sodium HA and is mediated by inhibiting HDAC5, unlike the anti-inflammatory mechanism of etanercept. These results demonstrate that SAP15 is useful as an inflammatory regulator of biomaterials and can be developed as a therapeutic for the treatment of inflammation.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Cell-Penetrating Peptides/pharmacology , Intracellular Space/drug effects , Protein Engineering , Amino Acid Sequence , Animals , Arthritis, Experimental/blood , Arthritis, Experimental/pathology , Body Weight/drug effects , Bone Resorption/diagnostic imaging , Bone Resorption/pathology , Cell-Penetrating Peptides/chemistry , Chondrocytes/drug effects , Female , Histone Deacetylases/metabolism , Humans , Inflammation/pathology , Mice , Organ Size/drug effects , Protein Structure, Secondary , RAW 264.7 Cells , Rats, Wistar , Signal Transduction/drug effects , X-Ray Microtomography
3.
Int J Mol Sci ; 21(12)2020 Jun 15.
Article in English | MEDLINE | ID: mdl-32549254

ABSTRACT

We report dual therapeutic effects of a synthetic heparin-binding peptide (HBP) corresponding to residues 15-24 of the heparin binding site in BMP4 in a collagen-induced rheumatic arthritis model (CIA) for the first time. The cell penetrating capacity of HBP led to improved cartilage recovery and anti-inflammatory effects via down-regulation of the iNOS-IFNγ-IL6 signaling pathway in inflamed RAW264.7 cells. Both arthritis and paw swelling scores were significantly improved following HBP injection into CIA model mice. Anti-rheumatic effects were accelerated upon combined treatment with Enbrel® and HBP. Serum IFNγ and IL6 concentrations were markedly reduced following intraperitoneal HBP injection in CIA mice. The anti-rheumatic effects of HBP in mice were similar to those of Enbrel®. Furthermore, the combination of Enbrel® and HBP induced similar anti-rheumatic and anti-inflammatory effects as Enbrel®. We further investigated the effect of HBP on damaged chondrocytes in CIA mice. Regenerative capacity of HBP was confirmed based on increased expression of chondrocyte biomarker genes, including aggrecan, collagen type II and TNFα, in adult human knee chondrocytes. These findings collectively support the utility of our cell-permeable bifunctional HBP with anti-inflammatory and chondrogenic properties as a potential source of therapeutic agents for degenerative inflammatory diseases.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , Bone Morphogenetic Protein 4/chemistry , Cell-Penetrating Peptides/administration & dosage , Heparin/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/metabolism , Binding Sites , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/drug effects , Chondrocytes/metabolism , Cytokines/blood , Disease Models, Animal , Drug Synergism , Etanercept/administration & dosage , Etanercept/pharmacology , Gene Expression Regulation/drug effects , Humans , Lipopolysaccharides/adverse effects , Male , Mice , RAW 264.7 Cells
4.
J Biomed Mater Res A ; 106(2): 531-542, 2018 02.
Article in English | MEDLINE | ID: mdl-28975732

ABSTRACT

Bioactive agents, including proteins and peptides, can be loaded into hydrogels to improve bone regenerative capacity with their controlled release. However, the current loading method has focused on physical mixing, which has limited release control. Therefore, alternative conjugation of bioactive agents with hydrogels is highly recommended. Direct chemical conjugation of synthetic peptides containing a functional moiety with a hydrogel would be ideal. Here, we synthesized a bioactive calcium accumulating peptide (CAP) containing a collagen binding motif, which can induce osteogenic differentiation. A tyrosine residue in CAP was used to directly chemically conjugate the peptide with a gelatin-based enzymatically crosslinked hydroxyphenyl propionic acid hydrogel under H2 O2 /Horse radish peroxidase conditions. To test the acceleration of bone formation, human periodontal ligament stem cells (PDLSCs) were loaded into a chemically conjugated CAP hydrogel. The CAP hydrogel induced bone mineralization around the PDLSCs and increased osteogenic marker expressions in vitro. It also recovered a bone layer in a calvarial defect 4 weeks postimplantation. In summary, an injectable CAP hydrogel scaffold system was developed as a potentially useful engineered microenvironment to enhance bone restoration, and it could be utilized as a vehicle for bioactive delivery of stem cells in tissue regenerative therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 531-542, 2018.


Subject(s)
Bone Regeneration/drug effects , Calcium/pharmacology , Gelatin/pharmacology , Hydrogels/pharmacology , Peptides/pharmacology , Amino Acid Sequence , Animals , Bone Morphogenetic Protein 2/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Humans , Osteocalcin/metabolism , Osteogenesis/drug effects , Osteopontin/chemistry , Peptides/chemical synthesis , Peptides/chemistry , Periodontal Ligament/cytology , Rats, Sprague-Dawley , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/ultrastructure
5.
Biochem Biophys Res Commun ; 491(3): 827-833, 2017 09 23.
Article in English | MEDLINE | ID: mdl-28554844

ABSTRACT

Cancer stem cells (CSCs) are a subpopulation of cancer cells and have been known to create cancer reoccurrence during cancer therapy due to their stem cell-like characteristics. However, exact target to control the CSC has not been fully established. Here, we enriched CD44High population of MDA-MB-231 cells by CD44 antibody as a CSC marker. By Phospho Antibody Array, CD44High population of MDA-MB-231 cells reveals Feline sarcoma-related tyrosine kinase (FER) protein was highly activated. When FER siRNA and low molecular weight protamine (LMWP) as cell penetrating peptides are applied to this population, cancer migration and colony forming ability are inhibited. Moreover, silencing FER using FER siRNA and LMWP conjugates enhances anti-metastasis related factors including E-cadherin, p75 and p63. Taken together, FER is a new marker for targeting breast CSCs and peptide-mediated siRNA method could be an effective and safe way of delivery and be a new therapeutic strategy for targeting breast cancer.


Subject(s)
Apoptosis/drug effects , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/physiology , Peptides/administration & dosage , Protein-Tyrosine Kinases/genetics , RNA, Small Interfering/therapeutic use , Apoptosis/genetics , Cell Line, Tumor , Gene Silencing , Gene Targeting/methods , Genetic Therapy/methods , Humans , Molecular Targeted Therapy/methods , Peptides/pharmacokinetics , Protein-Tyrosine Kinases/antagonists & inhibitors , RNA, Small Interfering/genetics , Treatment Outcome
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