Efficiency assessment of irrigation as an alternative method for improving the regenerative potential of non-healing wounds.

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult-to-heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non-healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long-term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre-defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non-invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

High Pressure 3 × 30 Minute Compression Methods for Advanced Lower Limb Lymphedema Patients.

Introduction: In advanced lymphedema of lower limbs, stage III bandaging under the routinely applied pressure of 40-60 mmHg remains largely ineffective. This is caused by skin and subcutaneous tissue stiffness due to fibrosis. Edema fluid accumulates deep in the subcutaneous tissue. Evacuating this fluid requires a high external compression force to overcome the resistance of fibrous tissue. We aimed to investigate the effectiveness of the compression method, with high pressure lasting for 3 days. Methods and Results: Twenty-one patients with lower limb lymphedema, stage III, of the postinflammatory type were included. Patients with acute inflammatory symptoms, venous thrombosis, profuse varicose veins, diabetes, and cardiac insufficiency with edema were excluded. A 10-cm-wide rubber bandage was applied to the foot and calf. The interface pressure measured using PicoPress ranged from 58 to 120 mmHg. Skin and deep tissue tonometry, skin water concentration, leg circumference, and drop of interface pressure were measured. Ultrasound examination was done before and after each compression session. The calf circumference decreased by 15.9 ± 5.4%, deep tissue stiffness by 58.9 ± 18.9%, skin stiffness by 69.6 ± 13.5%, and skin water concentration by 43.8 ± 11.5%. Interface pressure dropped to 66.3 mmHg (28-110 mmHg); ultrasonography images showed less fluid in the tissue. Conclusions: High-pressure 30-minute leg compression can remove excess edema fluid within 3 days and enable adjustment of nonstretch compression stockings. This method is more effective in advanced lymphedema at the beginning of therapy than the standard 30-50-mmHg bandaging as it provides an immediate effect.

Safety of GMP-compliant iPSC lines generated by Sendai virus transduction is dependent upon clone identity and sex of the donor.

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.

Lymphatic Vascular Insufficiency and Focal Edema in Early Stages of Noncancer-Related Lymphedema.

Introduction: Lymph flows along the lymphatics due to spontaneous contraction. However, injury and inflammation may deteriorate lymphatic' s endothelial and muscle cells and valves. In consequence, lymphatic vessels (LVs) become insufficient. Their contraction strength and rate slow down, and then lymph flow stops. Our study aimed to investigate the changes in lymph flow in early lymphedema cases. Methods and Results: In 36 patients with unilateral lymphedema stages 0 and I, we performed indocyanine green (ICG) lymphography, lymphoscintigraphy, skin water concentration, and stiffness measurement. We compared lymph flow velocity, LVs' appearance, contraction pattern, and rate between swollen and healthy limbs. ICG lymphography revealed (1) slower lymph flow after 3 minutes of foot movement; in lower calf level, lymphatics are seen in 22 (61.1%) swollen limbs compared with 36 (100%) healthy limbs (p < 0.0001); (2) dye spots in the foot (47.1%) and calves (13.9%) in swollen limbs; (3) dilated foot (41.7%) and calves' lymphatics (52.8%); (4) different patterns of lymphatics contractility with slower contractions rate and (5) higher fluorescent intensity in edema limbs. There was higher skin water concentration at foot and ankle level and higher skin stiffness in the foot. Conclusions: Our studies have shown the distortion in lymphatic function as dilatation, slower lymph flow, slower contraction rate, presence of areas with occluded lymphatics (dermal backflow in foot and calves-focal edema), and higher skin water concentration in these regions in limbs with early lymphedema. ICG lymphography can be used for the early detection of LV insufficiency, which allows early prophylactic implementation.

Lower Limb Lipedema-Superficial Lymph Flow, Skin Water Concentration, Skin and Subcutaneous Tissue Elasticity.

Background: Lipedema of lower limbs is characterized by bilateral accumulations of excess adipose tissue starting from the ankle to the hips and buttocks. The studies with lymphoscintigraphy (LSC) and magnetic resonance (MR) lymphography show altered transport index and enlarged lymphatic vessels (LVs). Our studies aimed to investigate the superficial lymph flow, water accumulation, skin and subcutaneous tissue elasticity, and the possibility of using this information to diagnose lipedema. Methods and Results: Fifty patients with lipedema and 50 control subjects (women) were included. The Indocyanine Green (ICG) lymphography, LSC, skin water measurement, skin durometry, and deep tissue tonometry were done in all participants. ICG lymphography revealed: (1) Slower lymph flow in lipedema patients; after 3 minutes of feet movement in a horizontal position, the ICG-dyed lymph reached the upper calf level in 8% of lipedema patients compared with 56% in the control group (p ˂ 0.0001). (2) More than three LVs were noticed more often in lipedema patients. (3) The higher number of abnormal LV images at all limb levels and during each observation stage with a statistically significant number of foggy and dilated. (4) Statistically significant higher fluorescent intensity in all limb levels. Skin water concentration was higher in the feet in lipedema (p = 0.000189). Conclusion: Our studies have shown the differences in superficial lymph flow and water concentration between lipedema and normal limbs. Data proove the usefulness of ICG lymphography, skin water concentration and skin and subcutaneous tissue elasticity measurements in diagnosing lipedema.

Secondary release of the peripheral nerve with autologous fat derivates benefits for functional and sensory recovery.

The reconstruction of nerve continuity after traumatic nerve injury is the gold standard in hand surgery. Immediate, tension-free, end-to-end nerve suture ensures the best prognosis. The recovery is mostly promising; however, in a few cases, insufficient outcomes in motor or sensory function are observed. Intra- and extra-fascicular scarring accompanies the nerve regeneration process and limits final outcomes. Secondary nerve release in those cases is recommended. Unfortunately, scarring recurrence cannot be eliminated after secondary revision and neurolysis. The supportive influences of mesenchymal stem cells in the process of nerve regeneration were observed in many preclinical studies. However, a limited number of studies in humans have analyzed the clinical usage of mesenchymal stem cells in peripheral nerve reconstruction and revisions. The objective of this study was to evaluate the effects of undifferentiated adipose-derived stromal/stem cell injection during a last-chance surgery (neurolysis, nerve release) on a previously reconstructed nerve. Three patients (one female, two males; mean age 59 ± 4.5 years at the time of injury), who experienced failure of reconstructions of median and ulnar nerves, were included in this study. During the revision surgery, nerve fascicles were released, and adipose-derived stromal/stem cells were administered through microinjections along the fascicles and around the adjacent tissues after external neurolysis. During 36 months of follow-up, patients noticed gradual signs of sensory and in consequence functional recovery. No adverse effects were observed. Simultaneous nerve release with adipose-derived stromal/stem cells support is a promising method in patients who need secondary nerve release after nerve reconstruction. This method can constitute an alternative procedure in patients experiencing recovery failure and allow improvement in cases of limited nerve regeneration. The study protocol was approved by the Institutional Review Board (IRB) at the Centre of Postgraduate Medical Education (No. 62/PB/2016) on September 14, 2016.