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Introduction: Wound healing is severely compromised in patients with diabetes owing to factors such poor blood circulation, delayed immune response, elevated blood sugar levels, and neuropathy. Although the development of new wound healing products and prevention of serious complications such as infections in wounds have received substantial interest, wound healing remains a challenge in regenerative medicine. Burn wounds, especially third-degree burns, are difficult to treat because they are associated with immune and inflammatory reactions and distributive shock. Wound care and treatment that protects the burn site from infection and allows wound healing can be achieved with bioengineered wound dressings. However, few studies have reported effective dressings for third-degree burn wounds, making it important to develop new dressing materials. Methods: In this study, we developed an artificial amniotic membrane (AM) using epithelial and mesenchymal cells derived from human amnion as a novel dressing material. The artificial AM was applied to the wound of a diabetic third-degree burn model and its wound healing ability was evaluated. Results: This artificial amnion produced multiple growth factors associated with angiogenesis, fibroblast proliferation, and anti-inflammation. In addition, angiogenesis and granulation tissue formation were promoted in the artificial AM-treated mouse group compared with the control group. Furthermore, the inflammatory phase was prolonged in the control group. Conclusions: Our preliminary results indicate that the artificial AM might be useful as a new dressing for refractory ulcers and third-degree burns. This artificial AM-based material represents great potential for downstream clinical research and treatment of diabetes patients with third-degree burns.

The formal verification of cryptographic protocols has been extensively studied in recent years. To verify the cryptographic protocol security, formal verification tools consider protocol properties as interactive processes involving a cryptographic functionality. In general, we formally define a cryptographic functionality as an abstract function. However, the actual cryptographic protocols used comprise complex combinations of cryptographic functionalities. Thus, we may not determine if the protocol is secure, even when the cryptographic protocol security with cryptographic functionalities is verified using verification tools. Increasing the reliability of the verification results necessitates the verification of the security properties of these algorithms using ProVerif. Many cryptographic algorithms have been designed as iterative algorithms. These include the Merkle and Damgård construction (MD construction) method for cryptographic hash functions and the cipher block chaining mode (CBC mode) for the block cipher mode of operation. However, formalizing an iterative execution is difficult in ProVerif. Thus, we propose a method for formalizing a model of iterative executions. In the proposed method, we describe to formalize iterative execution as a formal model of function calls. We demonstrate the validity of our proposed method by formally verifying the safety of the MD construction method, which behaves like an iterative execution by including a one-way compression function and internal variables changed by the output of these functions. We also present a method for formalizing a common block cipher mode of operation (i.e., CBC mode) used in the proposed method to handle iterative execution.

Abstract Background Prescribing with high levels of medical appropriateness and patient satisfaction improves adherence. However, its appropriateness does not always match patient preference. Deprescription is important for ensuring the safety of medication therapy, but is not straightforward. Although successful deprescribing requires knowledge of patients’ thoughts on their prescriptions and factors that influence their acceptance of deprescribing, few comprehensive studies have been conducted on this topic. The aim of this study was to identify factors that influence patients’ attitudes toward deprescribing and obtain hints on how to achieve higher patient satisfaction and prescribing adequacy. Methods A questionnaire was administered to hospitalized patients and a logistic regression analysis was conducted to examine factors that influence their attitude toward deprescribing. Individual factors affecting patients’ thoughts and wishes regarding prescribing were extracted and analysed in detail. Results The analysis included 106 patients, of whom 40 (37.7%) wished deprescribing. Logistic regression analysis showed that “Age”, “Wish to reduce the number and types of medications”, “Satisfaction”, “Concerns about side effects”, and “Wish not to have certain medications changed” were factors influencing attitudes toward deprescribing. The results suggested that the factors were influenced by patients’ perceptions and individual patient backgrounds. There was a gap between willingness to reduce medication and to change their medications. Seventy-eight percent of all respondents indicated that they would like to reduce the number and type of pills they take if possible. However, only 44.6% of these patients indicated that they would actually like to change their medication. Conclusions This study is the only one to comprehensively investigate prescription content, patient background, and patients’ thoughts on factors influencing attitudes toward deprescribing. This study revealed five factors that can influence inclination toward deprescribing. In addition, the results suggest that patients want to be able to feel well with fewer medications if possible. This information may be useful in determining prescriptions that have high validity and patient satisfaction. Further research is needed on the gap between willingness to reduce medications and to change medications.

Abstract Background Generic drugs are heavily promoted in Japan. The aim of this retrospective single-center study was to clarify whether the frequency and reason that patients request a switch from a generic drug to the original drug differ according to therapeutic category and dosage form. Methods This study was performed at Chiba University Hospital. Prescription inquiries about 121 generic drugs from community pharmacies over a 3-year period (from July 2014 to June 2017) were analyzed. Results Approximately 30% of the requests were related to the efficacy, safety, and comfort of the generic drug. The most cited motive was “patient’s desire with no reason given” at 44.5%. According to multiple logistic regression analysis, therapeutic categories and dosage forms were associated with the requests. The median request frequency differed according to therapeutic category and dosage form. The frequency was highest for “agents affecting the central nervous system” and “tablets and capsules”, respectively. Among the therapeutic categories, “agents affecting the central nervous system” had the highest median number of requests related to “decreased effectiveness”; “cardiovascular agents” had the highest median number of requests related to “physician’s instruction”; and “agents for the epidermis” had the highest median number of requests related to “uncomfortable to use”. Among dosage forms, the odds ratio for patients’ original drug request for “liniment and patch” was about 1.5 times that for “tablets and capsules”. “Liniment and patch” had the highest median frequency of requests related to “decreased effectiveness”, “uncomfortable to use”, and “patient’s desire with no reason given”. Conclusions The request frequency and reason differed according to therapeutic category and dosage form. Pharmacists should advise each patient properly about the choice and switching of drug brands, taking into account the therapeutic category and dosage form, especially liniments and patches.

Induced pluripotent stem cells (iPSCs) have become a prevalent topic after their discovery, advertised as an ethical alternative to embryonic stem cells (ESCs). Due to their ability to differentiate into several kinds of cells, including cardiomyocytes, researchers quickly realized the potential for differentiated cardiomyocytes to be used in the treatment of heart failure, a research area with few alternatives. This paper discusses the differentiation process for human iPSC-derived cardiomyocytes and the possible applications of said cells while answering some questions regarding ethical issues.

Engineered heart tissues (EHTs) that are fabricated using human induced pluripotent stem cells (hiPSCs) have been considered as potential cardiac tissue substitutes in case of heart failure. In the present study, we have created hiPSC-derived cardiac organoids (hiPSC-COs) comprised of hiPSC-derived cardiomyocytes, human umbilical vein endothelial cells, and human fibroblasts. To produce a beating conduit for patients suffering from congenital heart diseases, we constructed scaffold-free tubular EHTs (T-EHTs) using hiPSC-COs and bio-3D printing with needle arrays. The bio-3D printed T-EHTs were cut open and transplanted around the abdominal aorta as well as the inferior vena cava (IVC) of NOG mice. The transplanted T-EHTs were covered with the omentum, and the abdomen was closed after completion of the procedure. Additionally, to compare the functionality of hiPSC-COs with that of T-EHTs, we transplanted the former around the aorta and IVC as well as injecting them into the subcutaneous tissue on the back of the mice. After 1 m of the transplantation procedures, we observed the beating of the T-EHTs in the mice. In histological analysis, the T-EHTs showed clear striation of the myocardium and vascularization compared to hiPSC-COs transplanted around the aorta or in subcutaneous tissue. Based on these results, bio-3D-printed T-EHTs exhibited a better maturation in vivo as compared to the hiPSC-COs. Therefore, these beating T-EHTs may form conduits for congenital heart disease patients, and T-EHT transplantation can form a treatment option in such cases.

The development of tissue-engineered vascular grafts heavily relies on the availability of large animal models that allow long-term assessment of graft patency. Here Itoh et al. propose a novel model of immunodeficient pigs that allows long-term accommodation of human cell-derived three-dimensional bioprinted vascular tubes.

[This corrects the article DOI: 10.1371/journal.pone.0209162.].

[This corrects the article DOI: 10.1371/journal.pone.0230428.].

Cryopreservation is a method used for preserving living cells by cooling them to very low temperatures. Although cryopreservation methods for oocytes and embryos have been developed for use in reproductive medicine, there are no established methods yet for preserving cell aggregates (spheroids) in regenerative medicine. We have developed a bio-three-dimensional (3D) printer that can fabricate scaffold-free 3D constructs by loading spheroids onto a needle array. We fabricated several constructs such as blood vessels, liver, diaphragm, and a conduit for nerves by using this method. These constructs have the potential to be applied in patients. However, the process of fabricating tissue constructs (harvesting cells, expanding cells, making spheroids using cultured cells, printing constructs, and maturing constructs) is time-consuming. Therefore, cryopreservation methods for spheroids or constructs should be developed to increase the efficiency of this method for clinical use. Here, we developed a method for cryopreserving spheroids, which were then used to fabricate constructs. Fibroblast cell-based spheroids were cryopreserved in phosphate-buffered saline or cryopreservation solution at -80°C for 1 week. After thawing, spheroids in cryopreservation solution began to fuse on day 1. Cryopreserved spheroids were printed onto a needle array to fabricate a scaffold-free tubular construct using a bio-3D printer. After 7 days, the printed spheroids fused and formed scaffold-free constructs. We confirmed the viability of cells in the cryopreserved spheroids and fabricated tubular constructs. Our results indicate that spheroids can be cryopreserved and used to prepare scaffold-free constructs for clinical use.

A major challenge in cardiac tissue engineering is the host's immune response to artificial materials. To overcome this problem, we established a scaffold-free system for assembling cell constructs using an automated Bio-3D printer. This printer has previously been used to fabricate other three-dimensional (3D) constructs, including liver, blood vessels, and cartilage. In the present study, we tested the function in vivo of scaffold-free cardiac tubular construct fabricated using this system. Cardiomyocytes derived from induced pluripotent stem cells (iCells), endothelial cells, and fibroblasts were combined to make the spheroids. Subsequently, tubular cardiac constructs were fabricated by Bio-3D printer placing the spheroids on a needle array. Notably, the spheroid fusion and beat rate in the constructs were observed while still on the needle array. After removal from the needle array, electrical stimulation was used to test responsiveness of the constructs. An increased beat rate was observed during stimulation. Importantly, the constructs returned to their initial beat rate after stimulation was stopped. In addition, histological analysis shows cellular reorganization occurring in the cardiac constructs, which may mimic that observed during organ transplantation. Taken together, our results indicate that these engineered cardiac tubular constructs, which address both the limited supply of donor tissues as well as the immune-induced transplant rejection, has potential to be used for both clinical and drug testing applications. To our knowledge, this is the first time that cardiac tubular constructs have been produced using optimized Bio-3D printing technique and subsequently tested for their use as cardiac pumps.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.