Ultrastructural changes of capillaries of the rat exocrine pancreas at the late burn injury and the application of lyophilized porcine dermal xenograft

Глибокі поширені опіки характеризуються не лише пошкодженням покривних тканин, а й викликають морфологічні та функціональні зміни всіх органів і систем організму, які об’єднуються нозологічним поняттям «опікова хвороба». Біологічні медіатори запальної реакції та нітрозо-оксидативного стресу при термічному ураженні негативно впливають на стан внутрішніх органів, зокрема підшлункової залози. Для корекції опіків застосовують ксенотрансплантати, виготовлені на основі шкіри свині шляхом кріоконсервування у рідкому азоті з наступною ліофілізацією та подрібненням. Мета роботи – дослідити ультраструктурні зміни гемокапілярів екзокринної частини підшлункової залози щурів у пізні строки після опікової травми шкіри та за умов її корекції ліофілізованими трансплантатами. Матеріали і методи. 46 статевозрілих аутбредних щурів було поділено на три групи: І – інтактні тварини, ІІ – з опіковою травмою ІІб ступеня, ІІІ – тварини з опіковою травмою, яким проводили некректомію та покривали рану подрібненим субстратом ліофілізованої шкіри свині. На 14 та 21 доби проводили електронномікроскопічне дослідження гемокапілярів підшлункової залози та визначали еритроцитарний індекс інтоксикації, рівні та коефіцієнт середньомолекулярних пептидів в крові. Результати. У І групі тварин стінка кровоносних капілярів була утворена ендотеліоцитами витягнутої форми, які лежали на базальній мембрані, у просвіті поодинокі еритроцити. Ендотеліоцити містили ядро витягнутої форми, в каріоплазмі переважав еухроматин, гетерохроматин у вигляді невеликих грудок мав маргінальне розміщення. Внутрішня поверхня ендотелію утворювала вирости цитоплазматичної мембрани, які збільшували обмінну поверхню капілярної стінки. Ядра перицитів містили еухроматин. У ІІ групі змінювалась ультраструктура гемокапілярів – їх просвіти розширені та містили скупчення формених елементів крові. Локально спостерігалось пошкодження цілісності їх стінки. Цитоплазма ендотеліоцитів просвітлена, набрякла, значно пошкоджені органели, поодинокі піноцитозні пухирці. Ядра були ущільнені з переважанням гетерохроматину, пікнотичні, мали глибокі інвагінації каріолеми з погано вираженим перинуклеарним простором. Перицити містили електроннощільні ядра, їх цитоплазма мала гомогенний вигляд та погано виражені органели. У ІІІ групі тварин стан стінки гемокапілярів був близький до норми. Мембрани плазмолем ендотеліоцитів чітко контуровані з численними виростами на люменальній поверхні, цитоплазма дещо просвітлена, містила піноцитозні пухирці та незначно змінені органели. Ядра ендотеліоцитів великих розмірів, в каріоплазмі переважав еухроматин. Оптично щільні перицити тісно прилягали до базальної мембрани капілярів. За даними біохімічних досліджень опікова травма супроводжувалась розвитком синдрому ендотоксемії, про що свідчило зростання концентрацій середньомолекулярних пептидів та підвищення проникності еритроцитарних мембран. Висновки. У пізні строки після опікової травми шкіри у шурів розвиваються значні деструктивні зміни гемокапілярів екзокринної частини підшлункової залози, що проявляється пошкодженням компонентів ядер та цитоплазми ендотеліоцитів. При проведенні ранньої некректомії та застосуванні подрібненого субстрату шкіри свині на 14 та 21 добу встановлено зниження вмісту токсичних катаболітів у крові, що створює необхідні умови для відновлення компонентів стінки гемокапілярів і нормалізації трансендотеліального обміну.

Evaluation of in vitro biocompatibility of scaffolds for the repair of bone defects

The use of bone scaffolds in traumatology and orthopedics is an extremely important issue. The growing number of cases of significant bone defects, in particular after revision arthroplasty, combat trauma and due to the introduction of new methods of reconstructive surgery of bones and joints, requires more detailed studies of the using different osteoplastic materials. Materials and methods. As scaffolds used 4 types of materials that are most often used in the clinic for the correction of bone defects - ceramic hydroxylapatite, beta-tricalcium phosphate, allogeneic bone matrix treated with gamma irradiation, allogeneic bone matrix scaffold. The effect of matrices on the viability of normal human fibroblasts (M19 cell line) in cell culture in vitro was studied. The viability of cells after their co-cultivation with scaffolds was determined by colorimetric method by staining with crystal violet. To obtain an osteoinductive effect used platelet-rich plasma (PRP), standardized by the method of Araki with some modifications. The proliferative activity of fibroblasts was assessed by the level of expression of the proliferation marker Ki-67 by immunocytochemical analysis. Results. It was found that the least pronounced antiproliferative effect is shown by allogeneic bone matrix treated with gamma irradiation. Data on the complex effect of co-cultivation of fibroblasts with scaffolds in the presence of PRP on cell viability and proliferative activity were obtained. It was found that PRP improves the survival of fibroblasts by 15-30 % and increases their proliferative activity by 35-75 %. Delipidization of scaffold from allogeneic bone matrix, heat-treated by local bone bank technology, increased its biocompatibility with human fibroblasts. Conclusions. According to the results of a comparative analysis of the impact of different scaffolds on the viability of normal human fibroblasts, it was found that scaffolds from allogeneic bone matrix have the least pronounced antiproliferative effect. Platelet-rich plasma has been shown to improve fibroblast survival and increase their proliferative activity. Treatment with 70 % ethyl alcohol scaffold from allogeneic bone matrix, heat-treated by local bone bank technology, increased its biocompatibility with human fibroblasts.

The effects of fetal neural cell conditioned medium on cell proliferation in the rat brain after traumatic brain injury

Traumatic brain injury (TBI) is accompanied by an increase in the number of proliferating cells. However, the question of the nature, conditions of production and mechanisms of action of humoral factors secreted by fetal neural cells (FNCs) on reparative processes and neurogenesis in the brain after trauma and FNCs transplantation remains open. The purpose of the study was to establish the possibility of the influence of the conditioned medium of fetal neural cell cultures on the proliferative activity of Ki-67-positive cells in the cortex and subcortical structures of the rat brain after TBI. Materials and methods. TBI was simulated by dropping a metal cylinder on the rat’s head. Rats (E17-18) were used to obtain cultures of neural stem/progenitor cells. Conditioned media from cell cultures with high adhesive properties (HA-CM) and low adhesive properties (LA-CM) were used to treat the effects of experimental TBI in rats by intramuscular injection. The effect of conditioned media on the proliferative activity of Ki-67-positive cells in the cortex and subcortical structures of the brain after TBI was determined by immunohistochemical analysis using antibodies against Ki-67 protein. Results. Immunohistochemical analysis of the brain sections showed that on the 5th day after traumatic brain injury in rats there was a probable increase in the number of Ki-67-positive cells in the cortex, hippocampus and thalamus. It was found that the injection of HA-CM or LA-CM in animals with TBI increased the number of Ki-67-positive cells in the hippocampus compared with the TBI group and their value for the TBI+LA-CM group reached 59.6 ± 6.1, and for the TBI+HA-CM group – 47.2 ± 3.1 cells (p <0.05 compared with the TBI group). In the cortex and thalamus, the number of Ki-67-positive cells in contrast decreased compared with the group of animals with TBI and for the group TBI+LA-CM was 20.2 ± 1.6 and 12.0 ± 1.7, respectively, and for the group TBI+HA-CM – 25.3 ± 2.1 and 13.3 ± 1.3, respectively. Conclusions. The administration of LA-CM or HA-CM to animals with traumatic brain injury increases the number of Ki-67-positive cells in the hippocampus, possibly associated with increased neurogenesis, and decreases in the cortex and thalamus, which may be due to a weakening of reactive gliosis.

The clinical effectiveness of cryopreserved human amniotic membrane in diabetic foot syndrome

Diabetic foot syndrome with long-term unhealed wounds is the most common complication and cause of limb amputation in diabetes. The search for effective therapeutic agents and their inclusion in treatment protocols is a priority due to the increase in the number of cases of this socially significant disease and disability among the working population every year. The aim of the study is to evaluate the effectiveness of cryopreserved human amniotic membrane in the treatment of long-term non-healing wounds of the lower extremities in diabetic foot syndrome. Materials and methods. The pilot clinical study described 4 clinical cases of treatment of patients with diabetes mellitus type I and II (1 woman and 3 men aged 52 to 68 years) with long-term unhealed wounds of the limbs under standard therapy. After previous wound sanation the applications of the cryopreserved human amniotic membrane were performed. Once a week after the application, the dynamics of wound healing was assessed. Blood glucose levels were determined before amniotic membrane treatment and two hours after the procedure. Results. As a results of weekly applications of human amniotic membrane there was a gradual decrease in the area of the wound from the original size and increase the rate of healing. Thus, at the time of the second visit (after 7 days) the reduction in the area of the ulcer from the initial size in patient 1 was 33 %, patient 2 – 25 %, patient 3 – 33 % on the sole and patient 4 – 3 %, and the healing rate – 4.7 %, 3.6 %, 4.7 % and 0.43 % per day, respectively. The use of human amniotic membrane did not affect blood glucose levels when comparing values before application and two hours after the procedure. Regular follow-up visits of patients 3, 6, 9 and 12 months after the start of the study showed no recurrence of ulcers. Conclusions. It has been shown that the use of cryopreserved human amniotic membrane in patients with diabetes mellitus and diabetic foot syndrome with long-term unhealed wounds results in complete healing of ulcers with stable remission during the year of observation.

The effect of mesenchymal stromal cells of various origins on mortality and neurologic deficit in acute ischemia-reperfusion in rats

Stroke is a global epidemic issue and the second leading cause of death in the world and in Ukraine. According to official statistics, every year 100-110 thousand Ukrainians suffer acute cerebrovascular disorders. One third of such patients are of working age, up to 50 % will have a disability, and only one in ten will fully return to full life. So far, promising experimental data on the treatment of neurological dysfunction using mesenchymal stromal cells (MSCs) have been obtained. The aim of study is to compare the effect of MSCs of different origins on mortality and neurologic deficit in rats with acute cerebral ischemia-reperfusion injury (CIRI). Materials and methods. Transient bilateral 20-minute occlusion of internal carotid arteries was modeled in male Wistar rats aged 4 months and animals were injected intravenously with MSCs derived from human umbilical cord Wharton's-jelly (hWJ-MSC), human and rat adipose tissue. Other groups of experimental animals were injected intravenously with rat fetal fibroblasts and cell lysate from hWJ-MSC. The last group of rats received Citicoline at a dose of 250 mg/kg as a reference drug. Control animals were injected intravenously with normal saline. The cerebroprotective effect of therapy was assessed by mortality and neurologic deficit in rats on the McGraw's stroke index score. Results. After 12 hours of observation in the crucial period in the development of experimental acute cerebrovascular disorders with the administration of hWJ-MSC, mortality was only 10 % against 45 % of animals in the control group. The use of rat fetal fibroblasts reduced the mortality of animals compare to the control group by an average of 25 %. CIRI in rats caused severe neurologic deficits: paralysis, paresis, ptosis, circling behavior. On the 7th day of observation in the control group of animals, the mean score on the McGrow's stroke index indicated severe neurological disorders. On the 14th day of observation in this group of animals there was no complete recovery of lost central nervous system functions. Compared with the control group of animals, all the treatment agents for acute CIRI (MSCs of various origins, MSC's lysate and Citicoline) contributed to a significant regression of neurologic deficit. Conclusions. Thus, transplantation of human Wharton's jelly-derived MSCs and rat fetal fibroblasts reduced mortality and alleviated neurological symptoms in rats with experimental ischemic stroke. hWJ-MSC, rat fetal fibroblasts, and rat adipose-derived MSCs reduced the incidence of neurological disorders better than Citicoline, which was accompanied by a regression of neurologic deficit dynamics on the 14th day of follow-up. The ability of stem cells of different origins to reduce neurologic deficit indicates the feasibility of their use in experimental acute cerebral ischemia.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

Liver injury associated with acute respiratory distress syndrome and the prospects of mesenchymal stem cells therapy for liver failure

The pathogenesis of acute respiratory distress syndrome (ARDS) includes neutrophilic alveolitis, alteration of alveolar epithelium and endothelium, formation of hyaline membranes and microvascular thrombosis, which results in an acute hypoxemic respiratory failure. ARDS results in major structural and cellular changes in organs and organ systems. It causes liver dysfunction in critical patients through paracrine action of cytokines and other pro-inflammatory mediators as well as hypoxemia, oxidative stress, toxins and hypoperfusion. Coronavirus disease 2019 (COVID-19)-associated ARDS affects liver through the development of systemic inflammatory response syndrome and hypoxia as well as cytokine storm. Liver injury manifests itself as increased plasma levels of hepatic transaminases and cholestatic liver enzymes. Stem cell therapy is one of the promising modern methods for treating ARDS-induced liver failure. Many studies showed the ability of multipotent mesenchymal stem cells (MMSCs) to differentiate into functional hepatocyte-like cells, which were then successfully used for liver regeneration. MMSCs were proven to be able to prevent the apoptosis of hepatocytes, as well as have anti-fibrotic and anti-inflammatory activity which allows their successful use in the treatment of ARDS-induced liver injury.

Clinical cases of late adhesive intestinal obstruction after liver and kidney transplantation in children and surgical methods of their treatment

Abdominal adhesions are one of the most common complications in abdominal surgery. In 56-70 % it is the cause of small bowel obstruction, which often requires repeated surgery. To date, a few cases of late adhesive intestinal obstruction (LAIO) as a complication of organ transplantation in children have been described. The purpose of the study was to investigate clinical cases of LAIO in a 9-year-old child after liver transplantation, and in an 8-year-old child after kidney transplantation. Materials and methods. Clinical case I. A 62-day-old girl was treated with Kasai procedure before a liver transplantation, according to type III atresia of the biliary tract. Orthotopic liver transplantation of the left-lateral section from living related donor was performed at the age of 9 months. The surgeries were accompanied by damage to the mesothelium and trauma of the serous membrane, which led to the formation of intraabdominal adhesions. With the growth of the child there was a growth of abdominal organs and adhesions stretching. LAIO was diagnosed 8 years after first surgery. Clinical case II. The boy was diagnosed with hypoplastic dysplasia of the right kidney, left-side ureterohydronephrosis, resulting in chronic end-stage renal disease. Peritoneal dialysis was performed at the age of 6 years. The effect of dialysate on the mesothelium led to the formation of small intestine pseudo-diverticula. At the age of 7 years a bilateral nephrectomy and kidney transplantation from a deceased donor were performed. At the age of 8 LAIO was diagnosed. In both cases, children underwent adhesiolysis. The peritoneum was sutured from the lower and upper edges of the wound to the middle, so that a hole remained in the middle of the wound. A Nelaton catheter was inserted through the hole and 250 ml of sodium hyaluronate solution "Defensal" was injected into the left-lateral part, small pelvis and right lateral abdominal cavity. Then anterior abdominal wall was sutured. Results. The successful surgical treatment of LAIO in children using local adhesiolysis in combination with sodium hyaluronate was performed. The careful handling of vascular anastomoses and changes in organ topography were required during surgery. The purpose of the surgery on LAIO was not only to restore the chyme transport in the gastrointestinal tract, but also to release the small intestine from the adhesions with the normal anatomy and function preservation. The observation of children for 5 years indicate the effectiveness of intraabdominal application of sodium hyaluronate solution.

The efficacy of fetal neural cell aggregates and their combination with fetal liver stromal cells to reduce brain damage after intracerebral hemorrhage in rats

Patients with intracerebral hemorrhage have frequent complications and high mortality. There are currently no effective treatments for this disease. We investigated the effect of the use of cryopreserved aggregates of neural cells in combination with fetal liver stromal cells on the reduction of rat brain injury after intracerebral hemorrhage. Methods. Intracerebral hemorrhage (ICH) was modeled in rats by stereotactic administration of 0.2 U of collagenase type IV into the striatum. Neural cell obtained from brain and stromal cells (SCs) – from liver of rat fetuses of 15 dpc. The suspension of neural cell aggregates (NCAs) alone or in combination with fetal liver stromal cells was injected into the lateral ventricle. The level of lipid peroxidation was determined by the thiobarbituric acid test. The degree of brain cells injury after ICH was determined by the activity of lactate dehydrogenase in blood serum. To assess the intensity of adverse factors and the regenerative potential of different variants of cell therapy, the area of the lost striatum in the rat brain and the average distance from the border of the lesion to the nearest neurons were determined. Results. Combined transplantation of NCAs with fetal liver SCs in rats with ICH was found to reduce malonic dialdehyde concentration and lactate dehydrogenase activity more effectively than NCAs alone, indicating inhibition of lipid peroxidation and reduction of cell injury after intracerebral hemorrhage as a result of the addition of SCs. It was shown a significant decrease in the area of lost striatum in both experimental groups. The single administration of NCAs reduced the distance from the lesion border to the nearest neurons the most, indicating the best conditions for survival and/or regeneration of neurons close to the lesion compared to controls. Conclusions. Administration of NCAs, both alone and in combination with fetal liver SCs, reduces the intensity of oxidative stress, preserves the intact striatum tissue, and increases the number of neurons near the brain lesion in intracerebral hemorrhage in rats. The co-transplantation of fetal liver SCs helps to inhibit lipid peroxidation more effectively.

The effects of combined administration of human umbilical cord-derived multipotent mesenchymal stromal cells and melatonin or fibroblast growth factor-2 to aged mice with a toxic cuprizone model of demyelination

The effect of transplantation of umbilical cord-derived multipotent mesenchymal stromal cells (UC-MMSCs) to patients with demyelinating diseases depends on the age of the recipient and can change under the influence of hormones or growth factors. Purpose. To investigate the effect of exogenous melatonin and recombinant human fibroblast growth factor-2 (rhFGF-2) on the effects of UC-MMSCs transplanted into aged mice with an experimental model of multiple sclerosis. Material and methods. 129/Sv mice, 15-17 months old, received the neurotoxin cuprizone with food for 3 weeks. From the 10th day of the cuprizone diet, 5•105 UC-MMSCs were injected intravenously. From the 11th day they received melatonin at 600 p.m. or rhFGF-2. The behavioral parameters were evaluated in the open field test and rotarod test. In the brain, the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and the level of malondialdehyde (MDA) were assessed. Results. Cuprizone intake reduces the behavioral response in mice compared to the intact group. The transplantation of UC-MMSCs increases the number of rearings and muscle tone in mice. Melatonin injections enhance the effects of cells on these parameters, as well as increase the motor and emotional activity of animals. The injection of rhFGF-2 preserves the effect of cells on behavioral response and increases locomotor activity in mice. After the injection of UC-MMSCs with melatonin or rhFGF-2, the content of MDA in the brain decreases and the activity of antioxidant enzymes increases, this is more significant under the influence of melatonin. Conclusion. Exogenous melatonin and rhFGF-2 improve the effects of transplanted UC-MMSCs on behavioral responses and brain antioxidant defenses in aged mice with cuprizone diet. At the same time, the positive effect of the combination of cells with melatonin is more pronounced.