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BACKGROUND@#Eye irritation tests with animals have been conducted for a long time. However, the subjective decision to irritation, the anatomic/physiologic difference between species and humans, and ethical issues are crucial problems. Various research groups have paid attention to alternative testing methods. In these senses, we fabricated in vitro minicornea models with immortalized human corneal epithelial cells (iHCECs) and keratocytes (iHCKs) and used them for irritation tests. This study hypothesized that our mini-cornea model could present different viability tendencies according to test chemicals with different irritancy levels. @*METHODS@#Cells used in this study were characterized with cornea-specific markers by immunocytochemistry and western blot. To make a three-dimensional hemisphere construct like cornea stroma, we cultured iHCKs under modified culture conditions verified by matrix formation and total collagen content. iHCECs were seeded on the construct and cultured at an air–liquid interface. The model was treated with 2-phenoxyethanol, triton X-100, sodium lauryl sulfate, and benzalkonium chloride. @*RESULTS@#iHCECs and iHCKs presented their specific cell markers. In modifying the culture condition, the group treating ascorbic acid (200 lg/ml) presented an intact cellular matrix and included the highest collagen content; thus, we used this condition to fabricate the mini-cornea model. The model shows hemisphere shape and homogenous cell distributions in histological analysis. We observed different sensitivity tendencies by types of chemicals, and the model’s viability significantly decreased when the chemical concentration increased. @*CONCLUSION@#In this study, we performed and observed irritation tests using a tissue-engineered mini-cornea model and considered to apply as an alternative approach for animal tests.
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BACKGROUND@#The extracellular matrix (ECM) has many functions, such as segregating tissues, providing support, and regulating intercellular communication. Cartilage-derived ECM (CECM) can be prepared via consecutive processes of chemical decellularization and enzyme treatment. The purpose of this study was to improve and treat osteoarthritis (OA) using porcine knee articular CECM. @*METHODS@#We assessed the rheological characteristics and pH of CECM solutions. Furthermore, we determined the effects of CECM on cell proliferation and cytotoxicity in the chondrocytes of New Zealand rabbits. The inhibitory effect of CECM on tumor necrosis factor (TNF)-a-induced cellular apoptosis was assessed using New Zealand rabbit chondrocytes and human synoviocytes. Finally, we examined the in vivo effects of CECM on inflammation control and cartilage degradation in an experimental OA-induced rat model. The rat model of OA was established by injecting monosodium iodoacetate into the intra-articular knee joint. The rats were then injected with CECM solution. Inflammation control and cartilage degradation were assessed by measuring the serum levels of proinflammatory cytokines and C-telopeptide of type II collagen and performing a histomorphological analysis. @*RESULTS@#CECM was found to be biocompatible and non-immunogenic, and could improve cell proliferation without inducing a toxic reaction. CECM significantly reduced cellular apoptosis due to TNF-a, significantly improved the survival of cells in inflammatory environments, and exerted anti-inflammatory effects. @*CONCLUSION@#Our findings suggest that CECM is an appropriate injectable material that mediates OA-induced inflammation.
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BACKGROUND@#Extracellular vesicles (EVs) exhibit potential as functional biomolecules for tissue regeneration and immunomodulation as they play important roles in the physiological communication between cells. EV internal cargo contains miRNAs, proteins, lipids, and so on. Osteoarthritis (OA) is a common joint disease causing disability owing to impaired joint function and pain. EVs originating from animal cells and tissue matrices are also being considered for OA, in addition to research involving non-steroidal therapeutic agents. However, there are no studies on EVs from marine organisms. Hence, we focused on sea cucumber-derived EVs and conducted experiments to set up an extraction protocol and to demonstrate their efficacy to modulate the inflammatory environment. @*METHODS@#Sea cucumber extracellular matrices (SECMs) were prepared by a decellularization process. Lyophilized SECMs were treated with collagenase and filtered to isolate sea cucumber extracellular vesicles (SEVs). After isolation, we conducted physical characterization and cell activation studies including cytotoxicity, proliferation, and anti-inflammation effect assays. @*RESULTS@#The physical characterization results showed circular SEVs in the size range of 66–480 nm. These SEVs contained large amounts of protein cargo, infiltrated the synoviocyte membrane without damage, and had a suppressive effect on inflammatory cytokines. @*CONCLUSION@#This study established an extraction process for EVs from sea cucumber and reported the anti-inflammatory ability of SEVs. Isolated SEVs can be further utilized for tissue regeneration studies and can be compared to various marine or animal-derived EVs.
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BACKGROUND@#Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. @*METHODS@#The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2 days and 7 days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. @*RESULTS@#The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. @*CONCLUSION@#These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.
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BACKGROUND@#Extracellular vesicles (EVs) exhibit potential as functional biomolecules for tissue regeneration and immunomodulation as they play important roles in the physiological communication between cells. EV internal cargo contains miRNAs, proteins, lipids, and so on. Osteoarthritis (OA) is a common joint disease causing disability owing to impaired joint function and pain. EVs originating from animal cells and tissue matrices are also being considered for OA, in addition to research involving non-steroidal therapeutic agents. However, there are no studies on EVs from marine organisms. Hence, we focused on sea cucumber-derived EVs and conducted experiments to set up an extraction protocol and to demonstrate their efficacy to modulate the inflammatory environment. @*METHODS@#Sea cucumber extracellular matrices (SECMs) were prepared by a decellularization process. Lyophilized SECMs were treated with collagenase and filtered to isolate sea cucumber extracellular vesicles (SEVs). After isolation, we conducted physical characterization and cell activation studies including cytotoxicity, proliferation, and anti-inflammation effect assays. @*RESULTS@#The physical characterization results showed circular SEVs in the size range of 66–480 nm. These SEVs contained large amounts of protein cargo, infiltrated the synoviocyte membrane without damage, and had a suppressive effect on inflammatory cytokines. @*CONCLUSION@#This study established an extraction process for EVs from sea cucumber and reported the anti-inflammatory ability of SEVs. Isolated SEVs can be further utilized for tissue regeneration studies and can be compared to various marine or animal-derived EVs.
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BACKGROUND@#Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. @*METHODS@#The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2 days and 7 days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. @*RESULTS@#The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. @*CONCLUSION@#These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.
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BACKGROUND: The fabrication of microchannels in hydrogel can facilitate the perfusion of nutrients and oxygen, which leads to guidance cues for vasculogenesis. Microchannel patterning in biomimetic hydrogels is a challenging issue for tissue regeneration because of the inherent low formability of hydrogels in a complex configuration. We fabricated microchannels using wire network molding and immobilized the angiogenic factors in the hydrogel and evaluated the vasculogenesis in vitro and in vivo. METHODS: Microchannels were fabricated in a hyaluronic acid-based biomimetic hydrogel by using “wire network molding” technology. Substance P was immobilized in acrylated hyaluronic acid for angiogenic cues using Michael type addition reaction. In vitro and in vivo angiogenic activities of hydrogel with microchannels were evaluated. RESULTS: In vitro cell culture experiment shows that cell viability in two experimental biomimetic hydrogels (with microchannels and microchannels + SP) was higher than that of a biomimetic hydrogel without microchannels (bulk group). Evaluation on differentiation of human mesenchymal stem cells (hMSCs) in biomimetic hydrogels with fabricated microchannels shows that the differentiation of hMSC into endothelial cells was significantly increased compared with that of the bulk group. In vivo angiogenesis analysis shows that thin blood vessels of approximately 25–30 µm in diameter were observed in the microchannel group and microchannel + SP group, whereas not seen in the bulk group. CONCLUSION: The strategy of fabricating microchannels in a biomimetic hydrogel and simultaneously providing a chemical cue for angiogenesis is a promising formula for large-scale tissue regeneration.
Subject(s)
Humans , Angiogenesis Inducing Agents , Biomimetics , Blood Vessels , Cell Culture Techniques , Cell Survival , Cues , Endothelial Cells , Fungi , Hyaluronic Acid , Hydrogels , Hydrogels , In Vitro Techniques , Mesenchymal Stem Cells , Oxygen , Perfusion , Regeneration , Substance PABSTRACT
Scaffolds with cartilage-like environment and suitable physical properties are critical for tissue-engineered cartilage repair. In this study, decellularized porcine cartilage-derived extracellular matrix (ECM) was utilized to fabricate ECM scaffolds. Mechanically reinforced ECM scaffolds were developed by combining salt-leaching and crosslinking for cartilage repair. The developed scaffolds were investigated with respect to their physicochemical properties and their cartilage tissue formation ability. The mechanically reinforced ECM scaffold showed similar mechanical strength to that of synthetic PLGA scaffold and expressed higher levels of cartilage-specific markers compared to those expressed by the ECM scaffold prepared by simple freeze-drying. These results demonstrated that the physical properties of ECM-derived scaffolds could be influenced by fabrication method, which provides suitable environments for the growth of chondrocytes. By extension, this study suggests a promising approach of natural biomaterials in cartilage tissue engineering.
Subject(s)
Biocompatible Materials , Cartilage , Chondrocytes , Extracellular Matrix , Methods , Tissue EngineeringABSTRACT
The extracellular matrix (ECM) is known to provide instructive cues for cell attachment, proliferation, differentiation, and ultimately tissue regeneration. The use of decellularized ECM scaffolds for regenerative-medicine approaches is rapidly expanding. In this study, cartilage acellular matrix (CAM)-based bioink was developed to fabricate functional biomolecule-containing scaffolds. The CAM provides an adequate cartilage tissue–favorable environment for chondrogenic differentiation of cells. Conventional manufacturing techniques such as salt leaching, solvent casting, gas forming, and freeze drying when applied to CAM-based scaffolds cannot precisely control the scaffold geometry for mimicking tissue shape. As an alternative to the scaffold fabrication methods, 3D printing was recently introduced in the field of tissue engineering. 3D printing may better control the internal microstructure and external appearance because of the computer-assisted construction process. Hence, applications of the 3D printing technology to tissue engineering are rapidly proliferating. Therefore, printable ECM-based bioink should be developed for 3D structure stratification. The aim of this study was to develop printable natural CAM bioink for 3D printing of a tissue of irregular shape. Silk fibroin was chosen to support the printing of the CAM powder because it can be physically cross-linked and its viscosity can be easily controlled. The newly developed CAM-silk bioink was evaluated regarding printability, cell viability, and tissue differentiation. Moreover, we successfully demonstrated 3D printing of a cartilage-shaped scaffold using only this CAM-silk bioink. Future studies should assess the efficacy of in vivo implantation of 3D-printed cartilage-shaped scaffolds.
Subject(s)
Cartilage , Cell Survival , Cues , Extracellular Matrix , Fibroins , Freeze Drying , Printing, Three-Dimensional , Regeneration , Silk , Tissue Engineering , ViscosityABSTRACT
The need for organ and tissue regeneration in patients continues to increase because of a scarcity of donors, as well as biocompatibility issues in transplant immune rejection. To address this, scientists have investigated artificial tissues as an alternative to transplantation. Three-dimensional (3D) bioprinting technology is an additive manufacturing method that can be used for the fabrication of 3D functional tissues or organs. This technology promises to replicate the complex architecture of structures in natural tissue. To date, 3D bioprinting strategies have confirmed their potential practice in regenerative medicine to fabricate the transplantable hard tissues, including cartilage and bone. However, 3D bioprinting approaches still have unsolved challenges to realize 3D hard tissues. In this manuscript, the current technical development, challenges, and future prospects of 3D bioprinting for engineering hard tissues are reviewed.
Subject(s)
Humans , Bioprinting , Cartilage , Methods , Regeneration , Regenerative Medicine , Tissue Donors , Tissue EngineeringABSTRACT
Larger animal models, such as porcine, have been validated as appropriate models of the human disc with respect to biomechanics and biochemistry. They are advantageous for research as the models are relatively straightforward to prepare and easily obtainable for research to perform surgical techniques. The intention of this study was to quantitatively analyze gene expression for collagen and proteoglycan components of the extracellular matrix and for collagenase (MMP-1) in porcine discs of varying ages (Newborn; 2-3weeks, Mature; 6-9 month, Older; 2-3 years). In this study, we observed that the cell number and GAG (glycosaminoglycan) formation dramatically decreased with aging. Also, gene expression in the annulus fibrosus (AF) and nucleus pulposus (NP) cells changed with aging. The level of MMP-1 mRNA increased with age and both type I, II collagens decreased with age. The level of aggrecan mRNA was highest in the mature group and decreased significantly with aging. In the mature group, MMP-1 expression was minimal compared to the newborn group. In AF cells, type II collagen was expressed at a high level in the mature group with a higher level of aggrecan, when aged NP showed a decrease in type II collagen. The model of IVD degeneration in the porcine disc shows many changes in gene expression with age that have been previously documented for human and may serve as a model for studying changes in IVD metabolism with age. We concluded that the porcine model is excellent to test hypotheses related to disc degeneration while permitting time-course study in biologically active systems.