Antlerogenic stem cells extract accelerate chronic wound healing: a preliminary study.

BACKGROUND: Chronic wounds constitute a significant medical and social problem. Chronic wound treatment may be supported by various techniques, such as negative pressure therapy, phototherapy or stem cells therapy, yet most of those supporting therapies need more evidence to be used for standard wound care. Current study covers the use of sonicated Antlerogenic Stem Cells (ASC) extract on chronic wounds. METHODS: Study was performed on 20 dermatological patients with venous leg ulcers, divided into two groups - treated with and without ASC extract respectively. The area and circumference of the wounds during the follow-up visits were measured on the wound imprint. Dynamics of wound healing was determined and compared between control and study group; statistics includes changes in absolute values (wound area, circumference), as well as relative (percentage of wound decrease, circumference/area ratio) and their change in time. For the purpose of Ki-67 immunohistochemical staining, sections were sampled from the wound edge at distinct check-points during therapy. Results of both groups were compared with Student test or Mann-Whitney test, depending on results distribution. RESULTS: Besides Ki-67 expression, all tested wound healing parameters (including relative and absolute wound decrease and changes in circumference/area ratio) were statistically significant more favorable in experimental group. CONCLUSION: ASC extract significantly supported standard chronic wound treatment. Due to small population of study the results should be considered preliminary, yet promising for further research.

Effect of Physical Training on the Levels of Sex Hormones and the Expression of Their Receptors in Rats With Induced Mammary Cancer in Secondary Prevention Model - Preliminary Study.

BACKGROUND/AIM: Breast cancer is the most common malignant tumor among women worldwide. In previous work, we presented results of physical activity in primary prevention in a model of induced mammary gland cancer. In the present study, we assessed the influence of physical activity on sex hormone levels (estradiol and progesterone) and the expression of their receptors (ER, PR), as well as the level of apoptosis of tumor cells in secondary prevention. MATERIALS AND METHODS: Fifty 1-month-old female Sprague-Dawley rats received intraperitoneal injection of 180 mg/kg body weight of N-methyl-N-nitrosourea (MNU) for tumor induction. Three months after the administration of MNU, rats were divided into four groups: low-intensity, moderate-intensity, and high-intensity physical training groups (combined as PT) and a sedentary control (SC) group. Physical training was conducted 5 days per week with a three-position treadmill according to a precisely described protocol. The entire training was completed by 32 rats from which tissue and blood were collected for further analysis. Immunohistochemistry for ER and PR expression, terminal deoxynucleotidyl transferase dUTP nick-end labeling method for detection of apoptosis, and enzyme-linked fluorescent assay for detection of plasma hormone levels (estradiol and progesterone) were performed. Statistical analysis used p<0.05 as the significance level. RESULTS: Significantly stronger expression of ER and PR was found in the SC in comparison to the PT group (p=0.035 and p=0.036, respectively). No statistically significant differences were found in estradiol or progesterone concentrations between SC and PT groups. Apoptosis was non-significantly increased in the PT group in comparison with the SC group. Stronger apoptosis in the PT group correlated positively with the level of training intensity (r=0.35, p=0.05). CONCLUSION: Physical training may reduce ER and PR expression in breast cancer cells, and reduce cell sensitivity to pro-proliferative and anti-apoptotic effects of estrogens, ultimately leading to apoptosis.

The Impact of Exercise Training on Breast Cancer.

BACKGROUND/AIM: Physical exercise is increasingly considered by many authors to be a factor reducing the risk of cancer development and premature cancer-related death. Data indicate higher cure rates and longer times of survival in cancer patients who regularly exercise. MATERIALS AND METHODS: A total of 50 female Sprague-Dawley rats were used in the experiment. Animals at 1 month of age were intraperitoneally injected with N-methyl-N-nitrosourea. Three months following drug administration, rats underwent supervised physical training. The animals were divided into four groups: control untrained group and 3 groups trained with different intensities - i.e. low, moderate and high. Routine histopathological examination of tumors was performed and mitotic activity was assessed by immunohistochemical expression of the Ki-67 antigen. RESULTS: Ki-67 antigen expression was observed in all analyzed tumors. The increase in Ki-67 antigen expression correlated positively with the increase in training intensity. CONCLUSION: It can be assumed that low-intensity physical training is safe for patients with breast cancer. However, moderate- and high-intensity training may induce tumor cell proliferation worsening patients' prognosis.

Impact of Physical Training on Sex Hormones and Their Receptors During N-Methyl-N-nitrosourea-induced Carcinogenesis in Rats.

BACKGROUND/AIM: The risk of breast cancer is related to duration of exposure to sex hormones, especially estrogen. The aim of this study was to assess the impact of physical training (PT) on estrogen and progesterone levels and expression of their receptors during carcinogenesis induced by N-methyl-N-nitrosourea (MNU) in rats. MATERIALS AND METHODS: Fifty female Sprague-Dawley rats were intraperitoneally administered MNU and divided into four groups: low-, moderate-, and high-intensity PT, and no PT (control). Plasma levels of sex hormones and tissue expression of their receptors were quantified and statistically analyzed. RESULTS: In the group of rats subjected to PT, a significantly higher progesterone level was observed. The highest progesterone level was noted in the low-intensity PT group. An increase in apoptosis of MNU-induced tumor cells was also demonstrated in the PT groups. CONCLUSION: PT stimulates apoptosis of tumor cells without an increase in their proliferative activity. The increase in apoptosis of tumor cells correlates positively with the progesterone level.

Reconstruction of cranial cruciate ligament in rabbits using polyester implants saturated with PRP, antlerogenic stem cells MIC-1 and their homogenate.

AIM OF THE STUDY: The attempt to limit the negative effects of polyester implants on the articular cavity by using preparations containing growth factors. MATERIALS AND METHODS: Polyester implants used for the reconstruction of a rabbit's cranial cruciate ligament (CCL) were saturated with autogenic platelet-rich plasma (PRP), antlerogenic stem cells MIC-1 and their homogenate prior to the surgery. Six months after CCL reconstruction, morphological, and biochemical blood tests were carried out, including proteinogram and acute phase proteins. The knee joints were also examined macro- and microscopically. RESULTS: The results, compared to the control group, showed a favorable effect of the PRP and homogenate of antlerogenic cells on limiting the inflammation caused by the presence of polyester implant in the knee joint. The addition of growth factors caused covering the implant faster with the recipient's connective tissue, thus contributing to reducing the inflammatory reaction of the articular capsule to the presence of polyester. At the same time, no enhanced local or general reaction of the rabbit organism was observed to the presence of xenogenic antlerogenic stem cells MIC-1 homogenate which, like the PRP, may provide an easily available source of growth factors, increasingly often used in regenerative medicine. CONCLUSIONS: Applying antlerogenic stem cells, their homogenate or PRP increases the volume of connective tissue that surrounds and intertwines polyester CCL implant, separating it from synovial cavity environment.

Antlerogenic stem cells: molecular features and potential in rabbit bone regeneration.

AIM: (i) To assess the expression profiles of stem cell-associated markers including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and Cd9, (ii) analyze the nanotopography of the MIC-1 stem cells and (iii) evaluate the efficiency of live stem cell implants and stem cell culture derivatives on the regeneration of bone deficiencies in rabbit mandibles. MATERIALS AND METHODS: The expression profiles of stem cell-associated genes, including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and CD9 were assessed using reverse transcription polymerase chain reaction and flow cytometry. Nanotopography of the antlerogenic MIC-1 cell lineage was analyzed using atomic force microscopy. The effect of MIC-1 stem cells, their homogenate and supernatant on the regeneration of bone deficiencies in rabbit mandibles was evaluated using histological analysis. The effect of MIC-1 stem cells and stem cell-based derivatives on the immune responses of the animals was assessed by analyses of acute phase protein levels (haptoglobin and fibrinogen). RESULTS: We found that the MIC-1 cells isolated from the apical regions of growing antlers exhibited molecular features that were characteristics of pluripotent stem cells. Using atomic force microscopy, we determined the details of the cell surface morphologies with a particular emphasis on the patterns of formation of plasma extensions for interlinking adjacent cells. We also demonstrated that not only implanted stem cells but also cell homogenates and cell post-culture supernatants have potential in the regeneration of bone deficiencies in the rabbit mandible. CONCLUSIONS: Our findings indicate that the use of both antlerogenic stem cell implants and the preparations derived from the cells offer alternative approaches to those based on autologous stem cells in the biological stimulation of osteogenesis and in bone regeneration.

The effect of physical training on the N-methyl-N-nitrosourea-induced mammary carcinogenesis of Sprague-Dawley rats.

The impact of physical activity on carcinogenesis has been demonstrated in many studies. Taking into account the discrepant results of physical exercise on the cell proliferation and apoptosis of breast cancer, we aimed to examine the impact of physical training on N-methyl-N-nitrosourea-(MNU)-induced mammary carcinogenesis. Fifty female rats were divided into four groups according to the intensity of physical activity they undertook. The number of developed tumors, tumor volume, and histopathological diagnoses were noted. Apoptosis and cell proliferation were studied by the number of TUNEL-positive and Ki-67-expressing cells. We demonstrated a statistically significant decrease in the tumor number between all trained groups and the control group. The results were most pronounced in the group with a moderate intensity of training. Moreover, we showed a decrease in tumor volume as training intensity increased, though the differences were not statistically significant. The mean number of TUNEL-positive cancer cells was significantly higher in the training groups than in the control group. These data suggest that physical training, especially of moderate intensity, may alleviate MNU-induced mammary carcinogenesis. The results could suggest that physical exercise-induced apoptosis may be a protective mechanism.

Immunohistochemical characterization of N-methyl-N-nitrosourea-induced mammary tumours of Sprague-Dawley rats.

BACKGROUND: Single dose of N-methyl-N-nitrosourea (MNU) was shown to induce malignant tumours in susceptible rat strains. However, such tumours are not well-characterized. MATERIAL AND METHODS: We characterized MNU-induced tumours in Sprague-Dawley rats using ultrasonographic, radiographic and immunohistochemical (IHC) methods. RESULTS: In 27 rats, 41 tumours developed, appearing ultrasonographically as hypodense, non-homogenic areas with signal enhancement at their periphery. Out of these, 39 were of malignant epithelial origin, with an IHC phenotype closely-resembling that of human invasive ductal breast carcinoma. One case was diagnosed as carcinosarcoma. IHC analysis revealed that Ki-67 antigen expression correlated positively with tumour volume (r=0.40, p=0.0079). Moreover, tumours with α-smooth muscle actin in the tumour stroma were characterized by a higher proliferative rate as compared to those without its expression (p<0.05). CONCLUSION: This rat model of chemical carcinogenesis may be suitable for examining breast cancer development and progression.

Characteristics of MIC-1 antlerogenic stem cells and their effect on hair growth in rabbits.

AIM: We characterized growth factors produced by MIC-1 antlerogenic stem cells and attempted to apply those cells to stimulate hair growth in rabbits. MATERIALS AND METHODS: We evaluated the gene and protein expression of growth factors by immunocytochemical and molecular biology techniques in MIC-1 cells. An animal model was used to assess the effects of xenogenous stem cells on hair growth. In the experimental group, rabbits were intradermally injected with MIC-1 stem cells, whereas the control group rabbits were given vehicle-only. After 1, 2 and 4 weeks, skin specimen were collected for histological and immunohistochemical tests. RESULTS: MIC-1 antlerogenic stem cells express growth factors, as confirmed at the mRNA and protein levels. Histological and immunohistochemical analysis demonstrated an increase in the number of hair follicles, as well as the amount of secondary hair in the follicles, without an immune response in animals injected intradermally with MIC-1 cells, compared to animals receiving vehicle-alone. CONCLUSION: MIC-1 cells accelerated hair growth in rabbits due to the activation of cells responsible for the regulation of the hair growth cycle through growth factors. Additionally, the xenogenous cell implant did not induce immune response.

Experimental xenoimplantation of antlerogenic cells into mandibular bone lesions in rabbits: two-year follow-up.

Different types of cells require activation, and take part in annual, dynamic growth of deer antlers. Stem cells play the most important role in this process. This report shows the results of a two-year long observation of xenogenic implant of antlerogenic stem cells (cell line MIC-1). The cells were derived from growing antler of a deer (Cervus elaphus), seeded onto Spongostan and placed in postoperative lesions of mandibular bones of 15 experimental rabbits. The healing process observed in the implantation sites in all rabbits was normal, and no local inflammatory response was ever observed. Histological and immunohistochemical evaluations were performed after 1, 2, 6, 12 and 24 months, and confirmed the participation of xenogenic cells in the regeneration processes, as well as a lack of rejection of the implants. The deficiencies in the bones were replaced by newly formed, thick fibrous bone tissue that underwent mineralization and was later remodelled into lamellar bone. The results of the experiment with rabbits allow us to believe that antlerogenic cells could be used in reconstruction of bone tissues in other species as well.

Effect of melatonin on melanoma cells subjected to UVA and UVB radiation in In vitro studies.

Studies performed in vivo and in vitro have shown that exogenous melatonin (Mel) exerts oncostatic effects on melanoma cells. Although the protective effect of Mel on skin and cells exposed mainly to UVB has been documented, effects of Mel have not yet been examined on melanoma cells exposed to UVA. Our investigations aimed at examination of the effect of Mel alone (0, 10(-3), 10(-6) and 10(-9) M), and after its addition to the culture medium for 30 minutes before exposure of melanoma cells to UVA (15 J/cm(2)) or UVB (30 mJ/cm(2), 60 mJ/cm(2)). Viability of the cells was examined using the colorimetric sulphorhodamine B test. Mel added to the medium at concentrations of 10(-3)-10(-8) M was found to increase the number of melanoma cells as compared to the control (cells with no Mel) after 24 hours. Compared to exposed cells without Mel, at 10(-3) M Mel, melanoma cells exposed to 30 mJ/cm(2) UVB increased in number and at 10(-9) M increased the survival of those exposed to 60 mJ/cm(2) UVB. The cells exposed to UVA (15 J/cm(2)) were protected by Mel at 10(-6) and 10(-9) M. Physiological concentrations of Mel exerted no oncostatic effects on the BM cell line used here. In addition, the pharmacological Mel concentrations stimulated proliferation of the cells. Beyond doubt, we have confirmed that Mel represents a substance which protects cells from UVA and UVB action in in vitro experiments.

Effect of melatonin on human keratinocytes and fibroblasts subjected to UVA and UVB radiation In vitro.

Skin represents one of the extrapineal sites of melatonin (Mel) synthesis. In the skin Mel plays, for example, the role of an antioxidant which scavenges and inactivates free radicals arising due to UV irradiation. Although the protective effect of Mel on skin and cells irradiated mainly with UVB has been documented, to date no comparison has been made for the effects of Mel on cells exposed to UVA. Our study aimed at evaluating the effect of Mel (0, 10(-3), 10(-6) or 10(-9) M) added to culture medium 30 minutes before exposure of keratinocytes and fibroblasts to irradiation with UVA (15 J/cm(2)) and UVB (30 mJ/cm(2), 60 mJ/cm(2)). Viability of the cells was evaluated using sulphorhodamine (SRB) colorimetric test. Mel at 10(-3) M increased the number of surviving keratinocytes and at 10(-6) M increased the number of surviving fibroblasts exposed to UVB (30 mJ/cm(2), 60 mJ/cm(2)) as compared to cells exposed only to radiation. In addition, 10(-6) M protected keratinocytes exposed to the dose of 30 mJ/cm(2). Mel at 10(-3) M exerted a protective effect on both types of cells irradiated with UVA (15 J/cm(2)). As documented by our studies, Mel protects skin cells from the action of UVA and UVB. The protective effect of different Mel concentrations might result from variable expression of melatonin receptors.

Development of foreign body giant cells in response to implantation of Spongostan as a scaffold for cartilage tissue engineering.

Spongostan, a gelatinous haemostatic sponge, is used in surgery. Moreover, Spongostan may serve as a scaffold for proteins or cells implanted into defects. At the site of biomaterial implantation, foreign body giant cells (FBGCs) may develop which are responsible for Spongostan degradation. The purpose of the present study was to examine whether Spongostan may serve as a scaffold in allogenic grafting of chondrocytes developed from rabbit auricular cartilage. The obtained results indicate that Spongostan fulfils its function as a cell scaffold, induces no inflammatory reaction and involves development of foreign body giant cells which participate in the process of its degradation. Microscopic observation showed that FBGCs manifest presence of cytoplasmic projections and lysosomes, which participate in phagocytosis of the applied scaffold.

[The protective role of melatonin in the course of UV exposure]. / Ochronna rola melatoniny podczas dzialaniapromieniowania UV.

Melatonin (Mel) is a hormone synthesized mainly by the pineal gland. The principal function of Mel in the body involves the control of circadian and seasonal rhythms. Moreover, numerous reports document its anti-oxidative properties. Skin and eyes are particularly sensitive to the noxious influences exerted by UV exposure. The most dangerous radiation of the UVB (ultraviolet-B) and UVA (ultraviolet-A) range induces the formation of reactive oxygen species and thus stimulates the apoptosis of exposed cells. In numerous in vivo and in vitro studies, Mel produced in the skin and eye has been found to protect against the sequelae of UVB- and UVA-induced oxidative stress. In in vitro studies involving UVB irradiation of keratinocytes, fibroblasts, and leukocytes, Mel applied in both pharmacological (10(-3) and 10(-4 )M) and physiological doses (10(-7) and 10(-9) M) decreased the fraction of damaged cells. A similar pattern of Mel action at various doses of Mel probably reflected the presence of melatonin receptors (mainly MT1 receptors) in skin and eye cells. Moreover, intraperitoneally administered Mel or Mel applied to the skin before UVB exposure protects against the development of cataract and erythema, respectively. Thus only intracellular Mel may protect cells against the effects of UVB exposure. Although there are numerous reports describing the effects of UVA on cells of the skin and eye, no studies have described the anti-oxidative properties of Mel in relation to UVA-irradiated cells.

Experimental application of xenogenous antlerogenic cells in replacement of auricular cartilage in rabbits.

BACKGROUND: Regenerative medicine in the recent years aims at explaining involvement of stem cells in regenerative processes and applying this knowledge in fulfilling human needs to find new, more efficient therapeutic methods. Growing antlers constitute a model organ for examining regeneration processes of tissues because they are the only mammalian appendages capable of regeneration. The rate of growth of deer antlers makes them one of the quickest growing structures in mammals. The cells taking part in this process have a considerable proliferating potential. The aim of the study was to analyze the possibility of using xenogenous antlerogenic cells (AC) in regeneration of cartilaginous tissues in non-immunosuppressed animals. METHODS: We undertook to use a xenogenous implant consisting of cultured antlerogenic mesenchymal cells suspended in hemostatic sponge in the reconstruction of lesions of ear cartilage in nine rabbits. A surgical site was prepared half-way up the outer, dorsal part of the right ear. About 1 cm from the free edge of the ear, a centrally peduncled flap of skin and perichondrium was prepared, measuring 1.5 cm x 1.5 cm. The exposed cartilage was excised in an area of about 1 cm x 1 cm. In the operated rabbits, in the prepared perichondrial pocket, we placed a flake of Spongostan saturated with the suspension of AC. Xenogenous cell survival and regeneration abilities were determined by histologic, immunohistochemical, and electron microscopy analysis of the grafts. RESULTS: In each case, healing occurred properly and neither local inflammation, necrosis nor implant rejection was observed. The hyaline cartilage lesion was replaced by new fibrous cartilage. This is similar to the histologic process occurring in growing deer antlers. The histologic, immunohistochemical, and electron microscopy analysis demonstrated the presence (and thus possible participation) of exogenous cells in the reconstructive process. At the same time, the immune response was very weak, which was confirmed by immunohistochemical reactions. CONCLUSION: Implanted antlerogenic cells were not rejected and possibly took part in the reconstruction of missing sections of the scaffolding of the rabbits' ear cartilages (although the mechanism is yet unknown). Low immunogenicity of AC, simplicity, efficiency, and low costs of production of implant material are the benefits of this method. Further research should unequivocally answer the question whether the MIC-1 cells are or are not the long-sought-after ideal material for the reconstruction of cartilaginous tissue lesions in various species, including human.

Expression of metallothionein in renal tubules of rats exposed to acute and endurance exercise.

The induction of exercise-induced apoptosis in not actively involved in exercise organs, such as kidney could be a result of oxidative stress. Metallothionein (MT) exerts a protective effect in the cell against oxidative stress and apoptosis. We have previously demonstrated an increased incidence of apoptosis in distal tubular cells and collecting ducts in rat kidney after acute exercise. The present study was designed to test the hypothesis that MT may play a protective role in rat renal tubules against exercise-induced apoptosis after the acute exercise and regular training. Male Wistar rats were divided into control, acute exercised and 8-wk regularly trained groups. The kidneys were removed after a rest period of 6 h and 96 h. The ultrastructure of renal tubular cells was examined by electron microscopy. Apoptosis was detected in paraffin sections by the TUNEL technique. Expression of MT was examined by immunohistochemistry. The level of lipid peroxidation (thiobarbituric acid reactive substances - TBARS) was assayed in renal tissue homogenates. After acute exercise, the occurrence of apoptosis was restricted to distal tubules and collecting ducts of rat kidney, whereas the proximal tubules remained unaffected. The 8-wk training did not result in increased apoptosis in tubular cell. MT expression was confined exclusively to proximal tubules in all groups. However, it was significantly increased in acutely exercised animals, as compared to control and trained rats. After the 8-wk training, MT expression remained unaltered as compared to the control group. TBARS levels were significantly increased after acute exercise, while after regular training they remained unchanged. A significant correlation between TBARS level and MT expression was demonstrated. The findings could suggest a protective role of MT against oxidative stress and apoptosis in proximal tubular cells.

[The multifaceted effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in organisms, especially dentition changes]. / Wielokierunkowe dzialanie 2,3,7,8-tetrachlorodibenzo-p-dioksyny (TCDD) na organizm ze szczególnym uwzglednieniem zmian w uzebieniu.

Dioxins are by products of various technological processes in many branches of industry and products of the combustion of chlorine compounds at low temperatures. They include polychlorated dibenzo-p-dioxins and dibenzofurans. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most dangerous to the human organism and is a model substance to define the toxicity of particular dioxins in mixture. TCDD in small doses exerts a toxic influence on embryos and maturing persons. Dioxin exposure to animals and people causes changes in the immunological system, fetus failure, and defects in fertility and in internal organs and is carcinogenic. Knowledge in this area is rather well documented and has expanded. However, there is little information about dioxin's influence on dentition development. In this review, recent literature reports and the results of own investigations of the general biological effects of dioxins, especially for dentition, are presented.

The search for stem cells of the epithelium in pulmonary alveoli.

In recent years significant progress has been witnessed in the identification of stem cells, which have now also been identified in the lungs. The aim of this was to induce post-pneumonia alveolar regeneration to facilitate the identification of stem cells. The studies were performed on Buffalo strain rats. Pneumonia was induced in the animals by a sub-pleural injection of carragenin. On days 4, 5 and 10 of the experiment both the control and experimental animals received intraperitoneal injections of bromodeoxyuridine (BrdU). Twenty-four hours after the last BrdU injection the rats were sacrificed and samples of the lungs were taken for examination. In order to detect proliferating cells in the paraffin sections, BrdU incorporation was detected with monoclonal antibodies. In pilot experiments BrdU incorporation was demonstrated in individual alveolar cells of variable distribution and of variable intensity in the colour reaction. The results have confirmed the existence of stem cells in pulmonary alveoli but their closer characterisation requires further studies with other techniques to detect pulmonary stem cells.

Apoptotic changes in the myocardium in the course of experimentally-induced pleurisy.

The secreted proinflammatory interleukins IL-1, IL-6 and TNF in the course of experimentally-induced pleurisy can be the cause of pathological changes in the ultrastructure of cardiac muscle and of apoptosis. The pleurisy was induced in rats by means of carrageenin. The scraps of cardiac muscle obtained during the inflammatory reaction in the pleura were analysed by means of an electron microscope. The scraps were also stained with the TUNEL method in order to find the apoptotic foci. It was proved by the experiment that the inflammatory process affected mitochondria in the cardiomyocytes, enhanced collagen fibre synthesis and contributed to the formation of apoptotic foci in the cardiac muscle.