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1.
Tissue Eng Regen Med ; 21(1): 97-109, 2024 01.
Article in English | MEDLINE | ID: mdl-38079100

ABSTRACT

BACKGROUND: Collagen is a key component of connective tissue and has been frequently used in the fabrication of medical devices for tissue regeneration. Human-originated collagen is particularly appealing due to its low immune response as an allograft biomaterial compared to xenografts and its ability to accelerate the regeneration process. Ethically and economically, adipose tissues available from liposuction clinics are a good resource to obtain human collagen. However, studies are still scarce on the extraction and characterization of human collagen, which originates from adipose tissue. The aim of this study is to establish a novel and simple method to extract collagen from human adipose tissue, characterize the collagen, and compare it with commercial-grade porcine collagen for tissue engineering applications. METHODS: We developed a method to extract the collagen from human adipose tissue under quasi-Good Manufacturing Practice (GMP) conditions, including freezing the tissue, blood removal, and ethanol-based purification. Various techniques, including protein quantification, decellularization assessment, SDS-PAGE, FTIR, and CD spectroscopy analysis, were used for characterization. Amino acid composition was compared with commercial collagen. Biocompatibility and cell proliferation tests were performed, and in vitro tests using collagen sponge scaffolds were conducted with statistical analysis. RESULTS: Our results showed that this human adipose-derived collagen was equivalent in quality to commercially available porcine collagen. In vitro testing demonstrated high cell attachment and the promotion of cell proliferation. CONCLUSION: In conclusion, we developed a simple and novel method to extract and characterize collagen and extracellular matrix from human adipose tissue, offering a potential alternative to animal-derived collagen for xeno-free tissue engineering applications.


Subject(s)
Tissue Engineering , Tissue Scaffolds , Swine , Animals , Humans , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Adipose Tissue/metabolism , Collagen/chemistry , Extracellular Matrix
2.
Dev Biol ; 461(2): 145-159, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32061586

ABSTRACT

scarecrow (scro) gene encodes a Drosophila homolog of mammalian Nkx2.1 that belongs to an evolutionally conserved NK2 family. Nkx2.1 has been well known for its role in the development of hypothalamus, lung, thyroid gland, and brain. However, little is known about biological roles of scro. To understand scro functions, we generated two types of knock-in mutant alleles, substituting part of either exon-2 or exon-3 for EGFP (or Gal4) by employing the CRISPR/Cas9 genome editing tool. Using these mutations, we characterized spatio-temporal expression patterns of the scro gene and its mutant phenotypes. Homozygous knock-in mutants are lethal during embryonic and early larval development. In developing embryos, scro is exclusively expressed in the pharyngeal primordia and numerous neural clusters in the central nervous system (CNS). In postembryonic stages, the most prominent scro expression is detected in the larval and adult optic lobes, suggesting that scro plays a role for the development and/or function of this tissue type. Notch signaling is the earliest factor known to act for the development of the optic lobe. scro mutants lacked mitotic cells and Delta expression in the optic anlagen, and showed altered expression of several proneural and neurogenic genes including Delta and Notch. Furthermore, scro mutants showed grossly deformed neuroepithelial (NE) cells in the developing optic lobe and severely malformed adult optic lobes, the phenotypes of which are shown in Notch or Delta mutants, suggesting scro acting epistatic to the Notch signaling. From these data together, we propose that scro plays an essential role for the development of the optic lobe, possibly acting as a regional specification factor.


Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/genetics , Homeodomain Proteins/physiology , Optic Lobe, Nonmammalian/embryology , Alleles , Animals , Brain/growth & development , CRISPR-Cas Systems , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/growth & development , Embryo, Nonmammalian , Exons/genetics , Gene Editing , Gene Expression Regulation, Developmental , Gene Knock-In Techniques , Genes, Reporter , Homeodomain Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Larva , Membrane Proteins/physiology , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Optic Lobe, Nonmammalian/growth & development , Receptors, Notch/physiology
3.
Gene ; 604: 9-21, 2017 Mar 10.
Article in English | MEDLINE | ID: mdl-27940109

ABSTRACT

Although the mechanisms of apoptotic cell death have been well studied in the fruit fly, Drosophila melanogaster, it is unclear whether such mechanisms are conserved in other distantly related species. Using degenerate primers and PCR, we cloned a proapoptotic gene homologous to Head involution defective (Hid) from the Scuttle fly, Megaselia scalaris (MsHid). MsHid cDNA encodes a 197-amino acid-long polypeptide, which so far is the smallest HID protein. PCR analyses revealed that the MsHid gene consists of four exons and three introns. Ectopic expression of MsHid in various peptidergic neurons and non-neuronal tissues in Drosophila effectively induced apoptosis of these cells. However, deletion of either conserved domain, N-terminal IBM or C-terminal MTS, abolished the apoptogenic activity of MsHID, indicating that these two domains are indispensable. Expression of MsHid was found in all life stages, but more prominently in embryos and pupae. MsHid is actively expressed in the central nervous system (CNS), indicating its important role in CNS development. Together MsHID is likely to be an important cell death inducer during embryonic and post-embryonic development in this species. In addition, we found 2-fold induction of MsHid expression in UV-irradiated embryos, indicating a possible role for MsHid in UV-induced apoptosis.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Diptera/genetics , Drosophila Proteins/genetics , Gene Expression Regulation, Developmental , Neuropeptides/genetics , Amino Acid Sequence , Animals , Apoptosis/genetics , Apoptosis/radiation effects , Apoptosis Regulatory Proteins/metabolism , Base Sequence , Cloning, Molecular , Conserved Sequence , Diptera/growth & development , Diptera/metabolism , Diptera/radiation effects , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Embryo, Nonmammalian , Exons , Introns , Larva/genetics , Larva/growth & development , Larva/metabolism , Larva/radiation effects , Neuropeptides/metabolism , Plasmids/chemistry , Plasmids/metabolism , Pupa/genetics , Pupa/growth & development , Pupa/metabolism , Pupa/radiation effects , Sequence Homology, Amino Acid , Ultraviolet Rays
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