Injectable and Multifunctional Hydrogels Based on Poly(N-acryloyl glycinamide) and Alginate Derivatives for Antitumor Drug Delivery.

Chemotherapy is a conventional treatment that uses drugs to kill cancer cells; however, it may induce side effects and may be incompletely effective, leading to the risk of tumor recurrence. To address this issue, we developed novel injectable thermal/near-infrared (NIR)-responsive hydrogels to control drug release. The injectable hydrogel formulation was composed of biocompatible alginates, poly(N-acryloyl glycinamide) (PNAGA) copolymers with an upper critical solution temperature, and NIR-responsive cross-linkers containing coumarin groups, which were gelated through bioorthogonal inverse electron demand Diels-Alder reactions. The hydrogels exhibited quick gelation times (120-800 s) and high drug loading efficiencies (>90%). The hydrogels demonstrated a higher percentage of drug release at 37 °C than that at 25 °C due to the enhanced swelling behavior of temperature-responsive PNAGA moieties. Upon NIR irradiation, the hydrogels released most of the entrapped doxorubicin (DOX) (97%) owing to the cleavage of NIR-sensitive coumarin ester groups. The hydrogels displayed biocompatibility with normal cells, while induced antitumor activity toward cancer cells. DOX/hydrogels treated with NIR light inhibited tumor growth in nude mice bearing tumors. In addition, the injected hydrogels emitted red fluorescence upon excitation at a green wavelength, so that the drug delivery and hydrogel degradation in vivo could be tracked in the xenograft model.

Reduction-Responsive Chitosan-Based Injectable Hydrogels for Enhanced Anticancer Therapy.

Selective delivery of anticancer drug molecules to the tumor site enhances local drug dosages, which leads to the death of cancer cells while simultaneously minimizing the negative effects of chemotherapy on other tissues, thereby improving the patient's quality of life. To address this need, we developed reduction-responsive chitosan-based injectable hydrogels via the inverse electron demand Diels-Alder reaction between tetrazine groups of disulfide-based cross-linkers and norbornene groups of chitosan derivatives, which were applied to the controlled delivery of doxorubicin (DOX). The swelling ratio, gelation time (90-500 s), mechanical strength (G'~350-850 Pa), network morphology, and drug-loading efficiency (≥92%) of developed hydrogels were investigated. The in vitro release studies of the DOX-loaded hydrogels were performed at pH 7.4 and 5.0 with and without DTT (10 mM). The biocompatibility of pure hydrogel and the in vitro anticancer activity of DOX-loaded hydrogels were demonstrated via MTT assay on HEK-293 and HT-29 cancer cell lines, respectively.

Tissue Engineered Mini-Cornea Model for Eye Irritation Test.

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 mini-cornea 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 µg/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.

Corneal Repair with Adhesive Cell Sheets of Fetal Cartilage-Derived Stem Cells.

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.

The Perimensical Capsule: Potential Supporting Structure Surrounding Meniscus.

This study analyzed the morphological and biomechanical characteristics of perimeniscal capsule in knee joint thus establishing the roles of these tissues. A total of 10 human cadaver knees were used in this study. Medial meniscus and the adjacently surrounding joint capsules were harvested then sectioned both axially and coronally, followed by scanning electron microscopy analysis. The medial meniscus (anterior, middle, posterior) and the adjacent perimeniscal capsules (superior, peripheral) were biomechanically assessed to ascertain the tensile modulus. Among the perimeniscal capsules, the peripherally located capsules were morphologically different from the superiorly located capsules: The peripheral perimeniscal capsule was thicker and showed circumferentially oriented fibers whereas the superior perimeniscal capsule fibers were thinner and arranged in vertical orientation. The peripheral capsule also yielded significantly greater tensile modulus compared with the superior capsule biomechanically. We conclude that depending on its anatomical location, the perimeniscal capsule consists of fibers of varying orientations. This may be important in maintaining the circumferential hoop tension of the meniscus especially in the presence of circumferentially oriented and thick peripheral capsule fibers, which coincidentally have higher tensile modulus.

A 3D-printed polycaprolactone/ß-tricalcium phosphate mandibular prosthesis: A pilot animal study.

OBJECTIVE: In this study, we assessed the effectiveness of a tonsil-derived mesenchymal stem cell (TMSC)-transplanted polycaprolactone/beta-tricalcium phosphate prosthesis (specifically designed for easier fixing and grafting with a single scaffold) on rabbit mandible osteogenesis. METHODS: The mandibles of 18 rabbits were exposed, and 10 × 8-mm bone defects were made. Two rabbits did not receive implants; four were reconstructed with the scaffold control (SC) (SC group); four were reconstructed with scaffolds soaked in peripheral blood (PB) (PB group); four were reconstructed with TMSC-transplanted scaffolds (TMSC group); and four were reconstructed with differentiated osteocyte-transplanted scaffolds (DOC) (DOC group). Each rabbit was sacrificed 12 weeks after surgery, and the area of new bone formation was investigated by mechanical testing, histology, and micro-computed tomography. RESULTS: More extended and denser new bone masses were observed in the TMSC and DOC groups, although fibrosis and vascular formation levels were similar in all groups, suggesting that the dual-structured scaffold alone provides a good environment for bone attachment and regeneration. The bone volumes of representative scaffolds from the SC, PB, TMSC, and DOC groups were 43.12, 48.35, 53.10, and 57.44% of the total volumes, respectively. CONCLUSION: The design of the scaffold resulted in effective osteogenesis, and TMSCs showed osteogenic potency, indicating that their combination could enable effective bone regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 130:358-366, 2020.

Cross-linked cartilage acellular matrix film decreases postsurgical peritendinous adhesions.

Cartilage extracellular matrix contains antiadhesive and antiangiogenic molecules such as chondromodulin-1, thrombospondin-1, and endostatin. We have aimed to develop a cross-linked cartilage acellular matrix (CAM) barrier for peritendinous adhesion prevention. CAM film was fabricated using decellularized porcine cartilage tissue powder and chemical cross-linking. Biochemical analysis of the film showed retention of collagen and glycosaminoglycans after the fabrication process. Physical characterization of the film showed denser collagen microstructure, increased water contact angle, and higher tensile strength after cross-linking. The degradation time in vivo was 14 d after cross-linking. The film extract and film surface showed similar cell proliferation, while inhibiting cell migration and cell adhesion compared to standard media and culture plate, respectively. Application of the film after repair resulted in similar tendon healing and significantly less peritendinous adhesions in a rabbit Achilles tendon injury model compared to repair only group, demonstrated by histology, ultrasonography, and biomechanical testing. In conclusion, the current study developed a CAM film having biological properties of antiadhesion, together with biomechanical properties and degradation profile suitable for prevention of peritendinous adhesions.

Development of Printable Natural Cartilage Matrix Bioink for 3D Printing of Irregular Tissue Shape.

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.