Transplanted artificial amnion membrane enhanced wound healing in third-degree burn injury diabetic mouse model.

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.

Experiences and care needs of post-discharge patients who underwent cardiac surgery: A qualitative study.

AIM: This study clarifies the physical, psychological, and social forms of distress in, and care needs of, cardiac surgery patients, including optimal times for supporting them in their post-hospital discharge daily lives. METHODS: Semi-structured qualitative interviews were conducted. Participants included 12 adults (11 male and one female, mean age = 66.5 years) who had undergone cardiac surgery, experienced intensive care, and received outpatient care at the first post-discharge visit (around 2 ~ 3 weeks after discharge), around 3 months after discharge, and between 3 months and 1 year after discharge. Verbatim transcripts were analyzed based on similarities and differences for codes based on assessment items, and subcategories and categories were generated. RESULTS: After surgery, patients experienced physical, psychological, and social distress. First, they experienced physical pain shortly after discharge. Moreover, as they recovered at home, a gap between their sense of their recovery and the perceptions of those around them about their recovery often persisted, which led to psychological and social distress. Patients gained a sense of safety through "assurance of physical recovery" and security through "shared subjective distress." CONCLUSIONS: Post-cardiac surgery patients seek reassurance and safety by sharing experiences owing to daily life distress. Our findings could help provide better support to meet the care needs of such patients.

Inhibition of retinal ischemia-reperfusion injury in rats by inhalation of low-concentration hydrogen gas.

PURPOSE: To investigate the inhibitory effect of hydrogen gas inhalation on retinal ischemia reperfusion (I/R) injury using a rat model. METHODS: Six-week-old male Sprague-Dawley rats were used. A 27G needle connected by a tube to a saline bottle placed 200 cm above the eye was inserted into the anterior eye chamber to create a rat retinal I/R model. In the ischemia-plus-hydrogen-gas group (H2( +) group), the ischemia time was set to 90 min, and 1.8% hydrogen was added to the air delivered by the anesthesia mask simultaneously with the start of ischemia. In the non-hydrogen-treatment ischemia group (H2( -) group), I/R injury was created similarly, but only air was inhaled. ERGs were measured; after removal of the eyes, the retina was examined for histological, immunostaining, and molecular biological analyses. RESULTS: The mean thickness of the inner retinal layer in the H2( +) group was 107.2 ± 16.0 µm (n = 5), significantly greater than that in the H2( -) group (60.8 ± 6.7 µm). Immunostaining for Iba1 in the H2( -) group showed increased numbers of microglia and microglial infiltration into the subretinal space, while there was no increase in microglia in the H2( +) group. B-wave amplitudes in the H2( +) group were significantly higher than in the H2( -) group. In the membrane antibody array, levels of interleukin-6, monocyte chemotactic protein 1, and tumor necrosis factor alpha were significantly lower in the H2( +) group than in the H2( -) group. CONCLUSION: Inhalation of 1.8% hydrogen gas inhibited the induction of inflammation, morphological/structural changes, and glial cell increase caused by retinal I/R injury.

Hyperdry Human Amniotic Membrane as a Protective Dressing for Open Wounds With Exposed Bowel in Mice.

INTRODUCTION: An enteroatmospheric fistula forms when the exposed bowel is perforated with chronic enteric fistula formation. Currently, there is no established preventative method for this condition. Hyperdry (HD) amniotic membrane (AM) can promote early granulation tissue formation on the exposed viscera and is suitable for dressing intractable wounds as it possesses anti-inflammatory, antibacterial, and immunomodulatory properties. This study investigated whether HD-AM promotes early formation of blood vessel-containing granulation tissue for enteroatmospheric fistula treatment. METHODS: An experimental animal model of an open wound with exposed bowel was developed. A 15 × 20 mm wound was prepared on the abdomen of Institute of Cancer Research mice, and the HD-AM was placed. The mice were assigned to one of the following groups: HD-AM group, in which the stromal layer of the HD-AM was placed in contact with the exposed bowel; HD-AM UD group, in which the epithelial layer of the HD-AM was placed in contact with the exposed bowel; and the HD-AM (-) or control group, in which the HD-AM was not used. RESULTS: On postoperative days 7 and 14, granulation tissue thickness significantly increased in the HD-AM and HD-AM UD groups compared with that in the HD-AM (-) group. Macrophages accumulated in the HD-AM epithelium only in the HD-AM group. During HD-AM contact, a subset of invading macrophages switched from M1 to M2 phenotype. CONCLUSIONS: HD-AM is a practical wound dressing with its scaffolding function, regulation of TGF ß-1 and C-X-C motif chemokine 5 (CXCL-5), and ability to induce M1-to-M2 macrophage conversion.

[Autoimmune hemolytic anemia complicated with acute kidney injury and tubulopathy due to hemoglobin casts].

A 62-year-old male patient was admitted for close monitoring of anemia (hemoglobin level, 8.2 g/dl). Hemolytic anemia was observed; however, the direct antiglobulin test (DAT) result (standard tube method) was negative. Nevertheless, autoimmune hemolytic anemia (AIHA) was still suspected; therefore, a DAT (Colum method) and quantifying levels of red-blood-cell bound immunoglobulin G were performed, resulting in a definite diagnosis of warm AIHA. The patient also had an acute kidney injury (AKI) from the time of admission, which was poorly improved by supplemental fluids therapy alone. Therefore, renal biopsy was performed. Renal biopsy revealed acute tubular injury due to hemoglobin columns, and a diagnosed AKI caused by hemolysis due to AIHA. Following the definitive diagnosis of AIHA, the patient was treated with prednisolone, and after approximately 2 weeks, the anemia and nephropathy completely improved, which is maintained to this day. We report this case as a rare case of AKI induced by hemolysis of AIHA and a successful case of renal salvage by early administration of steroid.

Effect of a Community-Based Program to Accelerate Referral to Physicians for Individuals at High-Risk of Lifestyle-Related Diseases: A Cluster Randomized Trial.

AIM: A cluster randomized trial was conducted within 43 Japanese municipalities (21 intervention and 22 usual care) to examine whether the standardized health counseling for individuals at high cardiovascular risk screened at community sites accelerates clinic visits to strengthen the primary health care system. METHODS: Among high-risk individuals aged 40-74 years screened by health checkups, 8,977 and 6,733 were allocated to the intervention and usual care groups, respectively, who were not under medical treatment but had high levels of blood pressure (systolic/diastolic ≥ 160/100 mmHg), hemoglobin A1c or glucose (≥ 7.0% or corresponding glucose levels), LDL-cholesterol (≥ 180 mg/dL for men), and/or proteinuria of ≥ 2+. The intervention was performed from May 2014 to March 2016 under a standardized health counseling program based on the health belief model primarily by public health nurses. The usual care group was provided with local counseling protocols. RESULTS: The cumulative proportions of clinic visits for 12 months after health checkups were 58.1% (95% confidence interval, 57.0%, 59.3%) versus 44.5% (43.2%, 45.8%), with the probability ratio of clinic visits between the groups being 1.46 (1.24, 1.72). The between-group differences between the baseline and 1-year surveys were -1.50 (-2.59, -0.41) mmHg for diastolic blood pressure in the hypertension category, -0.30% (-0.53%, -0.07%) for HbA1c in the diabetes category, -0.37 (-0.48, -0.27) mmol/L for LDL-cholesterol in the dyslipidemia category, and none for proteinuria. CONCLUSION: Standardized health counseling for high-risk individuals accelerated clinic visits, with larger reductions in blood pressure, HbA1c, and LDL-cholesterol levels. The nationwide use of counseling after health checkups for high-risk individuals could help in controlling risk factors and in preventing lifestyle-related diseases.

Clinical Application of Hyperdry Amniotic Membrane in Cleft Palate Repair.

OBJECTIVE: To examine the safety and efficacy of hyperdry amniotic membrane (HDAM) for wound closure after palatoplasty in cleft palate patients. METHODS: HDAMs were prepared by washing and drying under infrared rays and microwaves at temperatures less than 60°C using a hyperdrying device. A total of 16 cleft palate patients (8 males, 8 females), aged 1 to 3 years (mean age 1 year 9 months), received one-stage pushback palatoplasty. The remaining raw wound after surgery was covered by an HDAM and a plastic cover plate. The cover plate was removed 1 week after surgery and parameters including temperature, feeding, allergic reactions, postoperative bleeding, re-epithelialization, wound dehiscence, and infection were monitored during the follow-up period of 31.2 months. RESULTS: All patients could adequately ingest at 5 days postoperation and after removal of the cover plate. None of the patients had a persistent fever or allergic reactions. Ingestion was feasible immediately in all patients, and no postoperative bleeding was observed during ingestion. No secondary hemorrhages were observed during follow-up. No postoperative wound dehiscence on the midline of the palate was observed. No infections were observed after the removal of the cover plate. No patients suffered from severe scar formation or contracture of the wound in the follow-up period. Hemorrhage, undue epithelialization, and scar contracture did not occur in any patient. The mean evaluation score was 7.75 points. CONCLUSION: HDAM can be used safely and effectively for wound closure following palatoplasty in cleft palate infants. Future studies testing the safety of patient's own amnion for palatoplasty, are required.

Importance of Housekeeping Gene Optimization for the Analysis of mRNA Expression During Wound Healing in a Third-Degree Burn Injury Model.

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.

Effect of hyperdry amniotic membrane in preventing tendon adhesion in a rabbit model.

BACKGROUND: No anti-adhesive materials are currently in clinical use for orthopaedic surgery. We developed a hyperdry amniotic membrane (HD-AM) for easy storage and transplantation as amniotic membrane. The purpose of this study was to examine the application of HD-AM to reduce peritendinous adhesions without impairing tendon healing. METHODS: We randomly divided 3 digits (2nd, 3rd, and 4th digits) from each rabbit into three groups: a tendon repair group; a tendon repair with HD-AM group (HD-AM group); and a control group (cast only). The effects of HD-AM on peritendinous adhesions and tendon healing were examined using microscopic, histological, and mechanical analyses in a rabbit flexor digitorum profundus tendon model. RESULTS: Adhesions on macroscopic evaluation of the tendon repair site were significantly smaller in the HD-AM group than in the tendon repair group. Little adhesion formation or foreign body reactions were seen by on histologic evaluation in the HD-AM group. Range of motion following tendon repair was significantly better in the HD-AM group than in the tendon repair group. Maximal tensile strength required to pull the tendon from the site of adhesion was significantly smaller in the HD-AM group than in the tendon repair group. As for tendon repair site, no significant difference was seen between the tendon repair and HD-AM groups. CONCLUSIONS: HD-AM prevented peritendinous adhesion macroscopically, pathologically, and mechanically without impairing the sutured tendon. HD-AM has already been clinically applied in neurosurgery, ophthalmology, and otolaryngology, and clinical application as an anti-adhesive materials may be achieved in the future.

Distribution of amniotic stem cells in human term amnion membrane.

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.

Identification of Chemo and Radio-Resistant Sub-Population of Stem Cells in Human Cervical Cancer HeLa Cells.

BACKGROUND: Cervical cancer ranks the second female malignancy after breast cancer. Cancer stem cells (CSCs) are hard to be eradicated, so can recur. We aim to isolate and characterize CSCs from HeLa cells. METHODS: These cells express clusters of differentiation (CDs), 44 and 24, to be sorted by fluorescence-activated cell sorting (FACS). RESULTS: CD44+CD24+ cells showed potential to form spheres, tumorigenicity, stemness genes and higher resistance to cisplatin, X-ray. CONCLUSION: CD44+CD24+ HeLa cells hold characteristics of CSCs, in vitro, in vivo studies, suggesting that targeting may lead to screening of new anti-cancer therapies.

What impact does postgraduate clinical training have on empathy among Japanese trainee dentists?

BACKGROUND: Enhancing empathy in healthcare education is a critical component in the development of a relationship between healthcare professionals and patients that would ensure better patient care; improved patient satisfaction, adherence to treatment, patients' medication self-efficacy, improved treatment outcomes, and reduced patient anxiety. Unfortunately, however, the decline of empathy among students has been frequently reported. It is especially common when the curriculum transitions to a clinical setting. However, some studies have questioned the significance and frequency of this decline. Thus, the purpose of this study was to determine the impact of postgraduate clinical training on dental trainees' empathy from cognitive, behavioral, and patients' perspective. METHODS: This study included 64 trainee dentists at Okayama University Hospital and 13 simulated patients (SPs). The trainee dentists carried out initial medical interviews with SPs twice, at the beginning and the end of their clinical training. The trainees completed the Japanese version of the Jefferson Scale of Empathy for health professionals just before each medical interview. The SPs evaluated the trainees' communication using an assessment questionnaire immediately after the medical interviews. The videotaped dialogue from the medical interviews was analyzed using the Roter Interaction Analysis System. RESULTS: No significant difference was found in the self-reported empathy score of trainees at the beginning and the end of the clinical training (107.73 [range, 85-134] vs. 108.34 [range, 69-138]; p = 0.643). Considering the results according to gender, male scored 104.06 (range, 88-118) vs. 101.06 (range, 71-122; p = 0.283) and female 109.17 (range, 85-134) vs. 111.20 (range, 69-138; p = 0.170). Similarly, there was no difference in the SPs' evaluation of trainees' communication (10.73 vs. 10.38, p = 0.434). Communication behavior in the emotional responsiveness category for trainees in the beginning was significantly higher than that at the end (2.47 vs. 1.14, p = 0.000). CONCLUSIONS: Overall, a one-year postgraduate dental training program neither reduced nor increased trainee dentists' empathy levels. Providing regular education support in this area may help trainees foster their empathy.

Hyperdry human amniotic membrane application as a wound dressing for a full-thickness skin excision after a third-degree burn injury.

BACKGROUND: Severe burn injuries create large skin defects that render the host susceptible to bacterial infections. Burn wound infection often causes systemic sepsis and severe septicemia, resulting in an increase in the mortality of patients with severe burn injuries. Therefore, appropriate wound care is important to prevent infection and improve patient outcomes. However, it is difficult to heal a third-degree burn injury. The aim of this study was to investigate whether hyperdry human amniotic membrane (HD-AM) could promote early granulation tissue formation after full-thickness skin excision in third-degree burn injury sites in mice. METHODS: After the development of HD-AM and creation of a third-degree burn injury model, the HD-AM was either placed or not placed on the wound area in the HD-AM group or HD-AM group, respectively. The groups were prepared for evaluation on postoperative days 1, 4 and 7. Azan staining was used for granulation tissue evaluation, and estimation of CD163, transforming growth factor beta-1 (TGF-ß1), vascular endothelial growth factor (VEGF), CD31, alpha-smooth muscle actin (α-SMA) and Iba1 expression was performed by immunohistochemical staining. Quantitative reverse-transcription polymerase chain reaction (PCR) was used to investigate gene expression of growth factors, cell migration chemokines and angiogenic and inflammatory markers. RESULTS: The HD-AM group showed significant early and qualitatively good growth of granulation tissue on the full-thickness skin excision site. HD-AM promoted early-phase inflammatory cell infiltration, fibroblast migration and angiogenesis in the granulation tissue. Additionally, the early infiltration of cells of the immune system was observed. CONCLUSIONS: HD-AM may be useful as a new wound dressing material for full-thickness skin excision sites after third-degree burn injuries, and may be a new therapeutic technique for improving the survival rate of patients with severe burn injuries.

Preconditioning of Adipose-Derived Mesenchymal Stem-Like Cells with Eugenol Potentiates Their Migration and Proliferation In Vitro and Therapeutic Abilities in Rat Hepatic Fibrosis.

Mesenchymal stem cells (MSCs) have considerable therapeutic abilities in various disorders, including hepatic fibrosis. They may be affected with different culture conditions. This study investigated, on molecular basics, the effect of pretreatment with eugenol on the characteristics of adipose tissue-derived MSCs (ASCs) in vitro and the implication of eugenol preconditioning on the in vivo therapeutic abilities of ASCs against CCl4-induced hepatic fibrosis in rats. The effect of eugenol on ASCs was assessed using viability, scratch migration and sphere formation assays. Expressions of genes and proteins were estimated by immunofluorescence or qRT-PCR. For the in vivo investigations, rats were divided into four groups: the normal control group, fibrotic (CCl4) group, CCl4+ASCs group and CCl4 + eugenol-preconditioned ASCs (CCl4+E-ASCs) group. Eugenol affected the viability of ASCs in a concentration- and time-dependent manner. Eugenol improved their self-renewal, proliferation and migration abilities and significantly increased their expression of c-Met, reduced expression 1 (Rex1), octamer-binding transcription factor 4 (Oct4) and nanog genes. Furthermore, E-ASCs showed more of a homing ability than ASCs and improved the serum levels of ALT, AST, albumin, total bilirubin and hyaluronic acid more efficient than ASCs in treating CCl4-induced hepatic fibrosis, which was confirmed with histopathology. More interestingly, compared to the CCl4+ASCs group, CCl4+E-ASCs group showed a lower expression of inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), cluster of differentiation 163 (CD163) and tumor necrosis factor-α (TNF-α) genes and higher expression of matrix metalloproteinase (MMP)-9 and MMP-13 genes. This study, for the first time, revealed that eugenol significantly improved the self-renewal, migration and proliferation characteristics of ASCs, in vitro. In addition, we demonstrated that eugenol-preconditioning significantly enhanced the therapeutic abilities of the injected ASCs against CCl4-induced hepatic fibrosis.

AT-MSCs Antifibrotic Activity is Improved by Eugenol through Modulation of TGF-ß/Smad Signaling Pathway in Rats.

For hepatic failure, stem cell transplantation has been chosen as an alternative therapy, especially for mesenchymal stem cells (MSCs). The aim of this study was to investigate the effect of eugenol (EUG) on the in vivo antifibrotic activity of adipose tissue-derived MSCs (AT-MSCs) and the underlying mechanism. After characterization of MSCs, rats were divided into five groups, Group 1 (normal control), Group 2 (CCl4), Group 3 (CCl4 + AT-MSCs), Group 4 (CCl4 + EUG) and Group 5 (CCl4 + AT-MSCs + EUG). Biochemical and histopathological investigations were performed. Furthermore, expression of type 1 collagen, α-SMA, TGF-ß1, Smad3 and P-Smad3 was estimated. Compared to the single treatment with AT-MSCs, the combination treatment of the fibrotic rats with AT-MSCs and EUG significantly improved the plasma fibrinogen concentration, IL-10 level and proliferating cell nuclear antigen expression, and also significantly decreased the serum levels of liver enzymes, IL-6, IL-1ß, TNF-α, type III collagen, hyaluronic acid, hydroxyproline and the TGF-ß growth factor. Furthermore, the combination treatment significantly decreased the hepatic expression of fibrotic markers genes (Type 1 collagen and α-SMA) and proteins (α-SMA, TGF-ß1 and phospho-Smad3) more than the treatment with AT-MSCs alone. We demonstrated that the combination treatment with EUG and AT-MSCs strongly inhibited the advancement of CCl4-induced hepatic fibrosis, compared with AT-MSCs alone, through TGF-ß/Smad pathway inhibition. This approach is completely novel, so more investigations are necessary to improve our perception of the underlying molecular mechanisms accountable for the effects of EUG on the antifibrotic potential of AT-MSCs.

Japan Trial in High-Risk Individuals to Enhance Their Referral to Physicians (J-HARP)-A Nurse-Led, Community-Based Prevention Program of Lifestyle-Related Disease.

BACKGROUND: It is uncertain whether health counselling after community-based health checkups for high-risk individuals of lifestyle-related disease enhances their referral to physicians. METHODS: We performed a clustered randomized controlled trial of untreated high-risk individuals aged 40 to 74 years who were screened from the annual health checkup in 2014 and 2015 under the national health insurance in 43 municipalities around Japan, assigning 21 intervention and 22 usual care municipalities. The high-risk conditions were severe forms of hypertension, diabetes, dyslipidemia (for men), and proteinuria. For the intervention group, the theory-based health counselling was performed to enhance referrals to physicians, while each municipality performed its own standard counselling for the usual care group. Data on clinical visits and risk factors were collected systematically and anonymously from the databases of health insurance qualification, health insurance claims, and annual health checkups. Hypotheses are that the cumulative proportion of seeing physicians (clinical visits) is higher in the intervention than the usual care groups, and that those in the intervention group have lower cumulative incidence of composite outcomes associated with lifestyle-related diseases. RESULTS: The numbers of subjects for the analyses were 8,977 in the intervention group and 6,733 in the usual care group. Among them, 6,758 had hypertension, 2,147 had diabetes, 2,861 had dyslipidemia, and 1,221 had proteinuria in the intervention group, with corresponding numbers of 4,833, 1,517, 2,262, and 845, respectively, in the usual care group. There were no material differences in mean levels and proportions of major cardiovascular risk factors between the two groups. CONCLUSIONS: We expect to provide scientific evidence on the effectiveness of health counselling.