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Purpose: Sodium taurocholate cotransporting polypeptide (NTCP) is a transmembrane protein responsible for delivering indocyanine green (ICG), an ideal infrared fluorescent dye, from extracellular space into the cytoplasm. Additionally, NTCP located in the hepatocyte membrane is the portal for hepatitis B and D virus (HBV/HDV) infections. This study verified the feasibility of NTCP as a reporter and further established a drug-screening platform for HBV/HDV infections.Procedures: NTCP was transduced into HT-29, a colorectal cancer cell line. To examine the use of NTCP as a reporter, NTCP-expressing cells were treated with ICG and examined through flow cytometry, an in vivo imaging system (IVIS), and confocal microscopy. Furthermore, ICG was administrated to NTCP-expressing tumor-bearing nude mice and examined using the IVIS. To study the drug-screening platform, NTCP-expressing cells were treated with cyclosporin A, an NTCP inhibitor, and ICG, and examined using a multimode detection platform. Moreover, nude mice were injected with NTCP inhibitors and ICG, and subsequently, their ICG signal was examined in vivo and in the blood.Results: In the reporter study, the ICG signal was higher in NTCP-expressing cells/tumors than in control cells/tumors after ICG treatment. In the drug-screening platform study, NTCP-expressing cells had decreased ICG intensity after treatment with NTCP inhibitors and ICG. Nude mice that were administered cyclosporin A had lower ICG intensity in the liver and higher intensity in the peripheral tissue and blood.Conclusions: NTCP and ICG form an ideal reporter system with extensive applications in cancer biology, robust drug-drug interactions, and drug screening in HBV/HDV infections. [ABSTRACT FROM AUTHOR]

Recent advances in tissue engineering have led to potential new strategies, especially decellularization protocols from natural tissues, for the repair, replacement, and regeneration of intervertebral discs. This study aimed to validate our previously reported method for the decellularization of annulus fibrosus (AF) tissue and to quantify potentially antigenic α-Gal epitopes in the decellularized tissue. Porcine AF tissue was decellularized using different freeze-thaw temperatures, chemical detergents, and incubation times in order to determine the optimal method for cell removal. The integrity of the decellularized material was determined using biochemical and mechanical tests. The α-Gal epitope was quantified before and after decellularization. Decellularization with freeze-thaw in liquid nitrogen, an ionic detergent (0.1% SDS), and a 24 h incubation period yielded the greatest retention of GAG and collagen relative to DNA reduction when tested as single variables. Combined, these optimal decellularization conditions preserved more GAG while removing the same amount of DNA as the conditions used in our previous study. Components and biomechanical properties of the AF matrix were retained. The decellularized AF scaffold exhibited suitable immune-compatibility, as evidenced by successful in vivo remodeling and a decrease in the α-Gal epitope. Our study defined the optimal conditions for decellularization of porcine AF tissues while preserving the biological composition and mechanical properties of the scaffold. Under these conditions, immunocompatibility was evidenced by successful in vivo remodeling and reduction of the α-Gal epitope in the decellularized material. Decellularized AF scaffolds are potential candidates for clinical applications in spinal surgery. [ABSTRACT FROM AUTHOR]

Background: Reconstruction of a segmental fracture with massive bone loss is still a challenge for orthopaedic surgeons. The aim of our study was to develop a suitable biodegradable thermosensitive hydrogel system as a carrier for bone morphogenetic protein (BMP)-2 delivery in the treatment of critical-sized femoral defects.Methods: A block copolymer composed of monomethoxypoly(ethylene glycol) (mPEG), poly(lactic-co-glycolic acid) (PLGA) and 2, 2'-Bis (2-oxazolin) (Box) was synthesized by ring opening polymerization. The synthesized block copolymer was characterized by (1)H-NMR spectroscopy and gel permeation chromatography (GPC). Different biophysical and biochemical properties of the synthesized copolymer, including temperature-induced structure changes, degradation rate, pH changes during hydrolytic degradation, cell toxicity, and the release profile of BMP-2, were also evaluated and/or were compared with those of a well-characterized mPEG-PLGA copolymer. In animal testing, rabbits (n = 36) that received critically sized (10 mm) femoral defects were divided into 6 groups. These experimental groups included an untreated group, autograft, and groups treated with the synthesized copolymer carrying different concentrations of BMP-2 (0, 5, 10, and 20 μg/ml). Bone repair was evaluated using X-ray radiography, histological staining, micro-computed tomography (μCT), biomarker examination and biomechanical testing in a 12-week treatment period.Results: A new thermosensitive mPEG-PLGA/Box/mPEG-PLGA block copolymer, or named as BOX copolymer, was successfully prepared. Compared to the reported mPEG-PLGA in vitro, the prepared BOX copolymer at the same weight percent concentrations exhibited wider temperature ranges of gelation, slower degradation rates, higher the pH values, as well as less cytotoxicity. Furthermore, the BMP-2 release from BOX hydrogel exhibited a near-linear release profile in vitro. In animal experiments, treatment of critical-sized bony defects with 25 wt% BOX hydrogel carrying BMP-2 effectively promoted fracture healing during the 12-week trial period and higher concentrations of BMP-2 treatment correlated with better bone quality. Most importantly, clinical outcome and bone healing in the BOX-hydrogel group with 20 μg/ml BMP-2 were nearly equivalent to those in the autograft group in a 12-week treatment course.Conclusion: These data support that the use of BOX hydrogel (25 wt%) as a drug delivery system is a promising method in the treatment of large bone defects. [ABSTRACT FROM AUTHOR]