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Wound healing evaluation methods in a third-degree burn injury model are categorized as histological (re-epithelialization and granulation tissue formation) and molecular (quantitative polymerase chain reaction). In general, mRNA expression is normalized to those of the housekeeping gene. Although the housekeeping gene expression is generally stable, it has been reported that the stability of these genes depends on the wound healing process and treatment method. In this study, we identified the most stable housekeeping gene (TATA-binding protein) for studying gene expression in a third-degree burn injury model, in which wound healing was promoted by grafting human amnion-derived mesenchymal cells. We investigated the wound healing effect of human amnion-derived mesenchymal cells in the injury model. The formation of granulation tissue, the differentiation from fibroblasts to myofibroblasts, and functional vascular structure were promoted in the full-thickness skin excision site by treatment with these cells. The expression of angiogenic, pro-inflammatory and anti-inflammatory related mRNA was measured and normalized to that of the housekeeping gene, showing that treatment with the cells promoted the infiltration of endothelial cells and differentiation of M1 and M2 macrophages. In conclusion, wound healing in a third-degree burn injury model can be accurately analyzed using the optimized housekeeping gene.

The absolute shortage of compatible liver donors and the growing number of potential recipients have led scientists to explore alternative approaches to providing tissue/ organ substitutes from bioengineered sources. Bioartificial regeneration of a fully functional tissue/organ replacement is highly dependent on the right combination of engineering tools, biological principles, and materiobiology horizons. Over the past two decades, remarkable achievements have been made in hepatic tissue engineering by converging various advanced interdisciplinary research approaches. Three-dimensional (3D) bioprinting has arisen as a promising state-of-the-art tool with strong potential to fabricate volumetric liver tissue/organ equivalents using viscosity- and degradation-controlled printable bioinks composed of hydrous microenvironments, and formulations containing living cells and associated supplements. Source of origin, biophysiochemical, or thermomechanical properties and crosslinking reaction kinetics are prerequisites for ideal bioink formulation and realizing the bioprinting process. In this review, we delve into the forecast of the potential future utility of bioprinting technology and the promise of tissue/organ-specific decellularized biomaterials as bioink substrates. Afterward, we outline various methods of decellularization, and the most relevant studies applying decellularized bioinks toward the bioengineering of in vitro liver models. Finally, the challenges and future prospects of decellularized material-based bioprinting in the direction of clinical regenerative medicine are presented to motivate further developments.

Although opioids are widely used to treat moderate to severe pain, opioid addiction and the opioid overdose epidemic are becoming more serious. Although opioid receptor antagonists/partial agonists, such as naltrexone and buprenorphine, have relatively low selectivity for the μ-opioid receptor (MOP), they have been used for the management of opioid use disorder. The utility of highly selective MOP antagonists remains to be evaluated. Here, we biologically and pharmacologically evaluated a novel nonpeptide ligand, UD-030, as a selective MOP antagonist. UD-030 had more than 100-fold higher binding affinity for the human MOP (Ki = 3.1 nM) than for δ-opioid, κ-opioid, and nociceptin receptors (Ki = 1800, 460, and 1800 nM, respectively) in competitive binding assays. The [35S]-GTPγS binding assay showed that UD-030 acts as a selective MOP full antagonist. The oral administration of UD-030 dose-dependently suppressed the acquisition and expression of morphine-induced conditioned place preference in C57BL/6J mice, and its effects were comparable to naltrexone. These results indicate the UD-030 may be a new candidate for the treatment of opioid use disorder, with characteristics that differ from traditional medications that are in clinical use.

Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane–derived stem cells. The amnion can be divided into a site that is continuous with the umbilical cord (region A), a site that adheres to the placenta (region B), and a site that is located opposite the placenta (region C). We found that human amnion epithelial stem cells (HAECs) that strongly express stem cell markers were abundant in area A. HAEC not only expressesed stem cell-specific surface markers TRA-1-60, Tra-1-81, SSEA4, SSEA3, but was also OCT-3/4 positive and had alkaline phosphatase activity. Human amniotic mesenchymal stem cells expressed KLF-A, OCTA, Oct3/4, c-MYC and Sox2 which is transcription factor. Especially, in regions A and B they have expressed CD73, and the higher expression of BCRP which is drug excretion transporter protein than the other parts. These data suggest that different types of stem cells may have existed in different area. The understanding the relation with characteristics of the stem cells in each area and function would allow for the efficient harvest of suitable HAE and HAM stem cells as using tool for regenerative medicine.

The fabrication of mature engineered cardiac tissue is one of the major challenges in cardiac tissue engineering. For this purpose, we attempted to apply the 3D bioprinting approach. Aiming to construct an oriented tissue, a fine fiber-shaped scaffold with a support structure was first designed using CAD software. Then, a 3D bioprinter and cell-adhesive bio-inks were utilized to fabricate this structure. The cell-adhesive bio-inks were synthesized by combining sodium alginate and gelatin with tyramine, respectively, to form pre-gel materials that allow enzymatic crosslinking by horseradish peroxidase. By absorbance measurements, we confirmed that the tyramine modification rate of each polymer was 0.535 mmol/g-alginate and 0.219 mmol/g-gelatin. The width of the fiber-shaped scaffold was 216.8 ± 24.3 μm for the fabricated scaffold, while the design value was 200 μm. After 3D printing and adhesion-adding treatment of the scaffold with these bio-ink materials, cardiomyocytes were seeded and cultured. As a result, the cells spread onto the scaffold, and the entire pre-tissue contracted synchronously by day 6 of culture, showing a greater pulsatility than in the early days. Video analysis showed that the beating rate of pre-myocardial tissue on day 6 was 31 beats/min. In addition, we confirmed that the cardiomyocytes partially elongated along the long axis of the fiber-shaped scaffold in the pre-tissue cultured for 15 days by staining actin, suggesting the possibility of cell orientation. Furthermore, treatment with adrenaline resulted in a 7.7-fold increase in peak beating rate compared to that before treatment (from 6 beats/min to 46 beats/min), confirming the responsiveness of the pre-tissues to the drug. These results indicate that 3D bioprinting effectively produces mature cultured myocardial tissue that is oriented, contracts synchronously, and is responsive to drugs.