Amnion bilayer for dressing and graft replacement for delayed grafting of full-thickness burns; A study in a rat model.

Burn is a common case in developing countries, with over half of fire-related deaths reported in Southeast Asia and full-thickness burns as a high mortality risk. Human amnion has been used as a wound dressing for centuries. In this study, a decellularised amnion overlaid with fibrin, "amnion bilayer (AB)," was used as a dressing immediately after burn and as a graft to replace the scar in Sprague-Dawley rats subjected to full-thickness burn model. The aim was to observe whether amnion bilayer can reduce damages in third-grade burn when skin replacement is deemed impossible. The burn was induced using an electrical solder, heated for 5 mins, and contacted on the rat's bare skin for 20 s. AB was applied as a (i) dressing immediately after induction and graft after eschar removal. Two groups (n = 6) were compared: AB and Sofra-Tulle ®, the National Hospital of Indonesia (NHI) protocol. Sections were stained with hematoxylin and eosin and Masson trichrome stains. Immunohistochemistry labelling was used to indicate scars (α-smooth muscle actin [α-SMA] and collagen-1) and angiogenesis (von Willebrand factor). Also, the macrophages inflammatory protein-3α (MIP-3α) indicates an early inflammatory process. The post dressing of the AB group demonstrated hair follicle remains and adipose tissue development. The NHI group appeared with a denatured matrix. Complete healing was seen in the AB group after 28 days with skin appendages similar to normal, while the NHI group showed no appendages in the centre of the actively inflamed area. The α-SMA was found in both groups. Collagen-1 was highly expressed in the NHI group, which led to a scar. Angiogenesis was found more in the AB group. The AB group had shown the capacity to accelerate complete healing and recover skin appendages better than the current protocol.

Transport viable heart tissue at physiological temperature yielded higher human cardiomyocytes compared to the conventional temperature.

This study investigated the optimum transport condition for heart tissue to recover single-cell cardiomyocytes for future in-vitro or in-vivo studies. The heart tissues were obtained from removing excessive myocardium discharged during the repair surgery of an excessive right atrial hypertrophy due to a congenital disease. The transportation temperature studied was the most used temperature (4 °C) or the conventional condition, compared to a physiological temperature(37 °C). The heart tissues were transported from the operating theatre to the lab maintained less than 30 min consistently. Single-cell isolation was enzymatically and mechanically performed using collagenase-V (160 U/mg) and proteinase-XXIV (7-14 U/mg) following the previously described protocol. The impact of temperature differences was observed by the density of cells harvested per mg tissue, cell viability, and the senescence signals, identified by the p21, p53 and caspase-9 mRNA expressions. Results the heart tissue transported at 37 °C yielded significantly higher viable cell density (p < 0.01) yielded viable cells significantly higher density (p < 0.01) than the 4 °C; 2,335 ± 849 cells per mg tissue, and 732 ± 425 cells per mg tissue, respectively. The percentage of viable cells in both groups showed no difference. Although the 37 °C group expressed the apoptosis genes such as p21, p53 and caspase9 by 2.5-, 5.41-, 5-fold respectively (p > 0.05). Nonetheless, the Nk×2.5 and MHC genes were expressed 1,7- and 3.56-fold higher than the 4 °C. and the c-Kit+ expression was 17.56-fold, however, statistically insignificant. Conclusion When needed for single-cell isolation, a heart tissue transported at 37 °C yielded higher cell density per mg tissue compared to at 4 °C, while other indicators of gene expressions for apoptosis, cardiac structural proteins, cardiac progenitor cells showed no difference. Further investigations of the isolated cells at different temperature conditions towards their proliferation and differentiation capacities in a 3-D scaffold would be essential.

Preparation of Cell-Seeded Heart Patch In Vitro; Co-Culture of Adipose-Derived Mesenchymal Stem Cell and Cardiomyocytes in Amnion Bilayer Patch.

INTRODUCTION: Cardiovascular disease is the second killer across the globe, while coronary disease is the major cause. Cell therapy is one alternative to regenerate the infarcted heart wall. MATERIALS AND METHODS: In this study, the cardiomyogenesis capacity of human adipose stem cells (hAdSC) and human cardiomyocytes (hCardio) cultured in a 3-D biological scaffold (decellularised amnion bilayer) for nine days in a static condition was investigated. The cardiomyogenesis capacity of hAdSC were identified using immunohistochemistry and RT-PCR. The population of the cells isolated from the heart tissue expressed cTnT-1 (13.38 ± 11.38%), cKit (7.85 ± 4.2%), ICAM (85.53 ± 8.69%), PECAM (61.63 ± 7.18%) and VCAM (35.9 ± 9.11%), while from the fat tissue expressed the mesenchymal phenotypes (CD73, CD90, CD105, but not CD45, CD34, CD11b, CD19 and HLA-DR). Two age groups of hAdSC donors were compared, the youngsters (30-40yo) and the elderly (60-70 yo). RESULTS: The co-culture showed that after 5-day incubation, the seeded graft in the hAdSC-30 group had a tube-like appearance while the hAdSC-60 group demonstrated a disorganised pattern, despite of the MSC expressions of the hAdSC-60 were significantly higher. Initial co-culture showed no difference of ATP counts among all groups, however the hAdSC-30 group had the highest ATP count after 9 days culture (p = 0.004). After normalising to the normal myocardium, only the hAdSC-60 group expressed cTnT and MHC, very low, seen during the initial cultivation, but then disappeared. Meanwhile, the hAdSC-30 group expressed α-actinin, MHC and cTnT in the Day-5. The PPAR also was higher in the Day-5 compared to the Day-9 (p < 0.005). CONCLUSION: Cardiomyogenesis capacity of hAdSC co-cultured with hCardio in a 3-D scaffold taken from the 30-40yo donor showed better morphology and viability than the 60-70yo group, but maintained less than 5 days in this system.

Characterisation of the single-cell human cardiomyocytes taken from the excess heart tissue of the right ventricular outlet in congenital heart disease.

Cardiovascular disease is the second highest cause of death across the globe. Myocardial infarction is one of the heart diseases that cause permanent impairment of the heart wall leads to heart failure. Cellular therapy might give hope to regenerate the damaged myocardium. Single cells isolated from an excess heart tissue obtained from the correction of the right ventricular hypertrophy in patients with Tetralogy of Fallot for future heart study were investigated. METHODS: Once resected, the heart tissues were transported at 37 °C, in Dulbecco's Modified Eagle's medium/ DMEM (4.5 g.L-1, antibiotic-antimycotic 3x, PRP10% (v/v)), to reach the lab within 30 min, weighted and grouped into less than 500 mg and more than 1000 mg (n = 4). Each sample was digested with 250 U.mL-1 Collagenase type V and 4U.mL-1 Proteinase XXIV in the MACS™ C-tube (Milltenyi, Germany), then dissociated using the MACS™ Octo Dissociator with Heater (Milltenyi, Germany) for 60 min at 37 °C. RESULTS: All cells isolated were rod-shaped cells; viability was up to 90%. The cell density obtained from the 500 mg group were 4,867 ± 899 cells.mg-1 tissue weight, significantly higher compared to the 1,000 mg group; had 557 ± 490 cells.mg-1 tissue weight (mean of (n = 3) ± 95% C.l). The isolated cells were analyzed using FACs BD Flowcytometer, expressed cTnT + 13.38%, PECAM-1 + /VCAM-1- 32.25%, cKit + 7.85%, ICAM + 85.53%, indicating the cardiomyocyte progenitor cells. CONCLUSION: Cardiomyocytes taken from the wasted heart tissue might be a candidate of cardiomyocytes source to study interventions to the heart as it contained up to 13.38% cardiomyocytes, and 32.25% of cardiac progenitor cells. Moreover, perhaps when cardiac cell therapy needs autologous cardiomyocytes, less than 500 mg tissue weight can be considered as sufficient.