Effect of macrophages combined with supernatant of mesenchymal stem cell culture and macrophage culture on wound healing in rats.

Wound healing is an orderly sequence of events restoring the integrity of the damaged tissue. It consists of inflammatory, proliferation, and remodeling phases. The objective of the current study was to investigate the effect of local transplantation of cultured macrophage loaded with mesenchymal stem cell/macrophage culture supernatants on wound healing. Sixty-four healthy adult male Wistar rats were randomized into 4 groups of sixteen animals each: 1) SHAM group. 2) MAC-MSC/SN group: One-milliliter application of a mixture comprising mesenchymal stem cell and macrophage culture supernatants in a 1:1 ratio was administered locally to the wound bed. 3) MAC group: Local transplantation of macrophage cells cultured in the wound bed. 4) MAC + MAC-MSC/SN group: Local transplantation of cultured macrophage in combination with mesenchymal stem cell/ macrophage culture supernatants in the wound bed. An incisional wound model was used for biomechanical studies, while an excisional wound model was used for biochemical, histopathological, and planimetric assessments. The wound area was significantly reduced in the MAC + MAC-MSC/SN group compared to other groups (P < 0.05). Biomechanical measurements from the MAC + MAC-MSC/SN group were significantly higher compared to other experimental groups (P < 0.05). Biochemical and quantitative histopathological analyses revealed a significant difference between MAC + MAC-MSC/SN and other groups (P < 0.05). MAC + MAC-MSC/SN showed the potential to improve wound healing significantly. This appears to work by angiogenesis stimulation, fibroblast proliferation, inflammation reduction, and granulation tissue formation during the initial stages of the healing process. This accelerated healing leads to earlier wound area reduction and enhanced tensile strength of the damaged area due to the reorganization of granulation tissue and collagen fibers.

Benefits of conditioned medium of nicotine-pulsed mesenchymal stem cells in experimental autoimmune hepatitis.

Previous data indicated that nicotine could modulate the immune regulatory potential of mesenchymal stem cells (MSCs). Currently, we intend to assess the effects of a conditioned medium of nicotine-pulsed mesenchymal stem cells in the experimental model of autoimmune hepatitis (AIH). Bone marrow-derived MSCs pulsed with 0,.1,.5, or 1 µM nicotine until the cells reached 90% confluency. Correspondent to in vitro results, the least effective concentration of nicotine that led to an anti-inflammatory environment by the MSC-conditioned medium was 0.5 µM. The murine model of AIH induced by Intravenous injection Concanavalin A (ConA). Mice were allocated to pretreatment (Concomitant treatment with ConA administration) or treatment groups and received un-pulsed MSC-conditioned medium (CM) or conditioned medium of nicotine (0.5 µM)-pulsed MSCs (CMN). The levels of ALT, AST, MPO, TNF-α, IFN-γ, and IL-6 were the highest in the ConA group than in the other groups. Pretreatment or treatment with the CMN caused a significant reduction in hepatic enzymes and inflammatory cytokines compared to pretreatment or treatment with CM. Both CM or CMN significantly decreased the numbers of activated TCD4+ and TCD8+ in the blood. More importantly, pre-treatment or treatment with CMN caused a better improvement in the histopathological appearance than pre-treatment or treatment with CM. The results of this study show that CMN rapidly controls the AIH mouse model, and therefore it may be considered as a new therapeutic approach for the treatment of AIH patients.

Evaluation of di-n-butyl phthalate reproductive toxicity in pregnant rats and their offspring and assessment of vitamin E administration in reducing toxicity.

Phthalates are environmental contaminants mostly used as plasticizers and additives in different products. Having endocrine-disrupting properties, phthalates are known as potential reproductive toxicants. The present study was conducted to evaluate the reproductive toxicity of di-n-butyl phthalate (DBP) in pregnant rats and their offspring and also to assess the ability of vitamin E in the elimination or reducing reproductive toxicity of DBP. Sixty-six pregnant Wistar rats were exposed to 100, 500 or 1,000 mg kg-1 per day DBP or 500 mg kg-1 per day DBP along with 100 mg kg-1 per day vitamin E during gestation. After delivery, they were divided into two groups. In one group gavage was finished after litter while in the other DBP administration was continued till weaning. The results showed that DBP affected many aspects of reproductive performance in pregnant rats and their offspring. It could be suggested that vitamin E could ameliorate the adverse effects of DBP, especially in male pups.

Evaluation of histopathological changes and exosomal biogenesis in pulmonary tissue of diabetic rats.

Diabetes mellitus is one of the leading causes of death globally. The development of cellular injuries and impaired energy metabolism are involved in the pathogenesis of diabetes mellitus, leading to severe diabetic complications in different tissues such as the pulmonary tissue. Autophagy is a double-edged sword mechanism required for maintaining cell survival and homeostasis. Any abnormalities in autophagic response can lead to the progression of several diseases. Here, we aimed to assess the effect of diabetic conditions on the autophagic response and exosome secretion in a rat model of type 2 diabetes mellitus. The experimental diabetic group received 45.00 mg kg-1 streptozocin (STZ) dissolved in 0.10 M sodium citrate. After 4 weeks, we monitored autophagic response and exosome biogenesis in the pulmonary tract using immunohistochemistry (IHC) and Real-time polymerase chain reaction analyses, respectively. Histological examination revealed the interstitial bronchopneumonia indicating enhanced immune cell infiltration into the pulmonary parenchyma. Immunohistochemistry staining displayed an enhanced autophagic response through the induction of microtuble-associated protein light chain 3 (LC3) and protein sequestosome 1 (P62) compared to the control rats. These changes coincided with significant induction of tetraspanin CD63 in STZ-induced diabetic rats relative to control rats. In conclusion, a diabetic condition can increase the autophagic response in pulmonary tissue. The accumulation of P62 in the pulmonary niche exhibits an incomplete autophagic response. The abnormal autophagy response can increase pulmonary cell sensitivity against injuries.

Angiogenic effects of cell therapy within a biomaterial scaffold in a rat hind limb ischemia model.

Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1-10% of humans worldwide with peripheral arterial disease. Cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs) have been used for the treatment of CLI. However, little information is available regarding the angiogenic potency of MSCs and mast cells (MC) in angiogenesis. The aim of this study was to evaluate the ability of MCs and MSCs to induce angiogenesis in a rat model of ischemic hind limb injury on a background of a tissue engineered hydrogel scaffold. Thirty rats were randomly divided into six control and experimental groups as follows: (a) Control healthy (b) Ischemic positive control with right femoral artery transection, (c) ischemia with hydrogel scaffold, (d) ischemia with hydrogel plus MSC, (e) ischemia with hydrogel plus MC and (f) ischemia with hydrogel plus MSC and MCs. 106 of each cell type, isolated from bone marrow stroma, was injected into the transected artery used to induce hind limb ischemia. The other hind limb served as a non-ischemic control. After 14 days, capillary density, vascular diameter, histomorphometry and immunohistochemistry at the transected location and in gastrocnemius muscles were evaluated. Capillary density and number of blood vessels in the region of the femoral artery transection in animals receiving MSCs and MCs was increased compared to control groups (P < 0.05). Generally the effect of MCs and MSCs was similar although the combined MC/MSC therapy resulted in a reduced, rather than enhanced, effect. In the gastrocnemius muscle, immunohistochemical and histomorphometric observation showed a great ratio of capillaries to muscle fibers in all the cell-receiving groups (P < 0.05). The data indicates that the combination of hydrogel and cell therapy generates a greater angiogenic potential at the ischemic site than cell therapy or hydrogels alone.

Mannosylated polylactic-co-glycolic acid (MN-PLGA) nanoparticles induce potent anti-tumor immunity in murine model of breast cancer.

Nanoparticle-based cancer immunotherapy is considered a novel and promising therapeutic strategy aimed at stimulating host immune responses against tumors. To this end, in the present study, mannan-decorated polylactic-co-glycolic acid (PLGA) nanoparticles containing tumor cell lysate (TCL) and poly riboinosinic polycytidylic acid (poly I:C) were used as antigen delivery systems to immunize breast tumor-bearing Balb/c mice. PLGA nanoparticles were fabricated employing a double emulsion solvent evaporation method. The formation of spherical and uniform nanoparticles (NPs) ranging 150-250 nm was detected by field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS). Four nanoformulation were used to treat mice and vaccination-induced immunological responses. Tumor regression and overall survival rate were evaluated in four experimental groups. Tumor cell lysate and poly I:C loaded mannan-decorated nanoparticles (TCL-Poly I:C) NP-MN caused a significant decrease in tumor growth and 2- to 3-fold improvement in survival times of the treated mice. The NPs with or without mannan decoration elicited stronger responses in terms of lymphocyte proliferation, delayed-type hypersensitivity and CD107a expression. Moreover, our data indicated that the production of IFN-γ and IL-2 increased while the production of IL-4 and IL-10 decreased in splenocytes culture supernatants. In the pathological evaluations, we found that necrosis and immune cells infiltration rate in the tumor tissue of the treated mice was elevated, while tumor cellularity and lung metastases significantly decreased in particular in the group that received (TCL-Poly I:C) NP-MN. Altogether, our findings suggested that the mannan-decorated PLGA NPs antigen delivery system had significant anti-tumor effects against the murine model of breast cancer and it could be considered as a step forward to human breast cancer immunotherapy.

Tenogenic effects of silymarin following experimental Achilles tendon transection in rats.

Tendon healing is prolonged due to the small number of cells, poor circulation, and low metabolism. The optimal tendon healing and its complete functional recovery have always been a challenge for researchers. Silymarin possesses anti-inflammatory, anti-oxidant, analgesic, and regenerative properties. The present study aimed to investigate the effects of silymarin on healing the Achilles tendon in rats. Twenty-four male Wistar rats were divided into two groups of control and treatment. After surgical preparation, a complete transverse incision was made in the middle part of the Achilles tendon, and then a modified Kessler suture was placed. The control group received 1.00 mL normal saline for five consecutive days, and the treatment group received 50.00 mg kg-1 of silymarin suspended in 1.00 mL normal saline for five days, orally. During the experimental period, Achilles functional index (AFI) was recorded. Six weeks after surgery, sampling was done. Histopathologically, a significant increase in the density of collagen fibers and reduction in neovascularization and inflammatory cells infiltration were observed in the treatment group. The biomechanical evaluation showed a significant increase in tensile strength of the tendon in the treatment group compared to the control group. The AFI results were concomitant with the results stated above, indicating an improvement in the AFI of rats in the treatment group. The present study results showed that oral administration of silymarin improved tissue healing indices, biomechanical properties, and functional index, leading to optimal healing of experimental Achilles tendon injury in the rat.

Histomorphometrical evaluation of extensor digitorum longus muscle in sciatic nerve regeneration using tissue engineering in rats.

Skeletal muscle atrophy induced by denervation is one of the common disorders in traumatic nerve injuries. The aim of this study was the evaluation of histomorphometrical changes of extensor digitorum longus muscle after denervation and its regeneration by tissue engineering. Ninety adult male Wistar rats were randomly divided into six main groups (n = 15) in three time periods (2, 4 and 8 weeks; n = 5). Control group was treated without surgery, in transection (Tr) group left sciatic nerve was transected, in scaffold (S) group only collagen gel scaffold was used, in mast cell (MC) group mast cells were used, mesenchymal stem cell (MSC) group was treated with mesenchymal stem cells and in MC+MSC group, mast cells along with mesenchymal stem cells were used. In the cellular groups, the scaffold and cells were mixed and placed in the transected nerve gap. The average diameter of muscle fibers, ratio of the muscle fibers nuclei to the fibrocytes nuclei (mn/fn), ratio of the muscle fibers nuclei number to the muscle fibers number (mn/mf), the average ratio of blood vessels to muscle fibers number (v/mf) and muscles weight in Tr group were the lowest compared to the other groups; but, in cellular and S groups, amelioration was observed according to the time period. However, in MC+MSC group, there were the highest ameliorative results. This study revealed that simultaneous use of MCs and MSCs mixed with collagen gel scaffold can be considered as a suitable approach to improve denervated skeletal muscle atrophy associated with sciatic nerve injury.

Histomorphometric and immunohistochemical evaluation of angiogenesis in ischemia by tissue engineering in rats: Role of mast cells.

The aim of this study was to find a proper method for improvement of ischemic condition in the rat hind limb and also to observe the efficacy of cell engraftment with alginate/gelatin three-dimensional scaffolds. Eighteen male Wistar rats weighing 200 to 250 g were randomly divided into three groups (n = 6) including a) ischemia group; in which femoral artery was removed after ligation at the distance of 5 mm, b) scaffold group; in which hydrogel scaffold was added to the site of transected femoral artery and c) test group; in which in addition to hydrogel scaffold, mast cells (MCs) were also added (1 × 106 cells). Analysis of capillary density, artery diameter, histomorphometric parameters and immunohistochemistry in transected location were done on day 14 after femoral artery transection. The average number of blood capillary was significantly higher in the test group than other groups. Also, the average number of medium and large blood vessels was significantly higher in the test group compared to ischemia and scaffold groups. Application of MCs through the use of hydrogel scaffolds (alginate/gelatin) can be considered as a new approach in the application of stem cells for therapeutic angiogenesis under ischemic conditions which can improve the angiogenesis process in patients with peripheral artery diseases.

Crocin exerts improving effects on indomethacin-induced small intestinal ulcer by antioxidant, anti-inflammatory and anti-apoptotic mechanisms.

Crocin is a plant-derived carotenoid and bears potent antioxidant property. Ranitidine (a histamine H2 receptor blocker) is used for peptic ulcer treatment. The present study was planned to investigate the effects of crocin and ranitidine on indomethacin-induced ulcer in small intestine of rats. Animals were randomized into two major groups including indo-methacin (10.00 mg kg-1, ulcer group, 48 rats) and normal saline (1.00 mL kg-1, intact group, 48 rats) groups. Each of these two major groups was subdivided into eight subgroups for intra-peritoneal (IP) injections of normal saline, crocin (2.50, 10.00 and 40.00 mg kg-1), ranitidine (5.00 and 20.00 mg kg-1), crocin (2.50 and 10.00 mg kg-1) plus ranitidine (5.00 mg kg-1). Indomethacin induced intestinal ulcer was characterized by bleeding, inflammation, epithelial hyperplasia and crypt loss. This non-steroidal anti-inflammatory drug (NSAID), indomethacin decreased goblet cell number and superoxide dismutase (SOD) activity and increased small intestine weight, organo-somatic index (OSI), malodealdehyde (MDA), tumor necrosis factor-α (TNF-α) and caspase-3 contents of intestine. Crocin resolved all the above-mentioned parameter changes induced by indomethacin. These treatments produced no significant effects on the above-mentioned parameters of intact group. The results of the present study showed tissue protective and anti-ulcer effects of crocin on small intestine by antioxidant, anti-inflammatory and anti-apoptotic mechanisms. Ranitidine alone showed no effect; however, in combination with crocin it exerted recovery effects. It is recommended that crocin, be considered as a therapeutic agent for NSAIDs-induced intestinal damage management.

Selenium-Enriched Saccharomyces cerevisiae Reduces the Progression of Colorectal Cancer.

Colorectal cancer is one of the most common causes of mortality in the world while malnutrition is responsible for one third of the problem. Selenium has been recommended for prevention of colorectal cancer. The present study was conducted to investigate the effect of selenium-enriched Saccharomyces cerevisiae in reducing colorectal cancer progression in rats. Five groups of 170-200-g weight rats (n = 40) including healthy and cancer controls, Saccharomyces cerevisiae, selenium, and selenium-enriched Saccharomyces cerevisiae-treated groups were examined. All animals except healthy control group received 40 mg 1,2-dimethylhydrazine (DMH) per kilogram weight of rat twice a week. The healthy group received normal saline, and synchronously, selenium group received soluble selenium (4 mg/mL), Saccharomyces cerevisiae and selenium-enriched groups received yeast with the density of 5 × 108 CFU/mL by daily gavage. All treatments were carried out for 5 weeks after the last injection. Animals were autopsied, and aberrant crypt foci (ACF) of ejected colon were studied in the 40th week. Microscopic sections were prepared for hematoxylin and eosin. Furthermore, immunohistochemical staining of CD31, BCL2, and P53 antibodies was performed. Macroscopic and microscopic evaluations showed that DMH had the least destructive effect in selenium-enriched Saccharomyces cerevisiae group compared to other groups. Selenium-enriched Saccharomyces cerevisiae reduces colorectal cancer progression by various mechanisms such as reduction in the number and size of ACF and alteration in the function of the proteins such as P53, BCL2, and CD31.

Mouse Bone Marrow-Derived Mast Cells Induce Angiogenesis by Tissue Engineering in Rats: Histological Evidence.

OBJECTIVES: Therapeutic angiogenesis is employed to induce vascular network formation and improve functional recovery in ischemia. The aim of this study is to find an appropriate method to recover local ischemic conditions. MATERIALS AND METHODS: In this experimental survey, 20 male Wistar rats weighing approximately 200-250 g were randomly divided into four experimental groups respectively: ischemia group in which the femoral artery was transected; phosphate buffer solution group (PBS) in which the femoral artery transected location was immersed with PBS; chitosan (CHIT) group in which the transected location was immersed in a 50 µL CHIT solution; and mast cell transplanted group in which the transected location was immersed with a mixture of 50 µL CHIT and 50 µL PBS that contained 1×106 mast cells. RESULTS: On day 14 after surgery, mean numbers of blood vessels of different sizes in the CHIT/mast cell group significantly increased compared to the other experimental groups (P<0.05). CONCLUSIONS: Our data suggest that mast cell reconstitution could offer a new approach for therapeutic angiogenesis in cases of peripheral arterial diseases.

Anti-atherosclerotic effect of Cynodon dactylon extract on experimentally induced hypercholesterolemia in rats.

Cynodon dactylon (Bermuda grass) is a perennial plant traditionally used as an herbal medicine in many countries. In the present study, anti-atherosclerotic property of ethanolic extract of C. dactylon was investigated in the experimentally induced hypercholesterolemia in rats. In this study, 36 male Wistar rats were selected and allocated into six groups (n = 6). The control group received a normal diet, sham group received a high cholesterol diet (HCD; 1.50% cholesterol and 24.00% fat) and other groups received a HCD and ethanolic extract of C. dactylon at low (100 mg kg-1), moderate (200 mg kg-1) and maximum (400 mg kg-1) doses via gavages. The last group received atorvastatin (10 mg kg-1) through gavage with a HCD. The study period for all groups was six months. At the end of this period, parameters including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were assessed in the blood samples. Additionally, histopathological and immunohistochemical examinations on coronary and aorta arteries sections were performed. The results showed an increase in vessels wall thickness and proliferation of smooth muscle cells in the HCD group, while these pathological changes were not seen in C. dactylon-treated groups. Treatment of HCD animals with C. dactylon positively changed lipid profile by lowering of TC, TG and LDL-C. The results indicate that C. dactylon prevents from early atherosclerotic changes in the vessels wall.

Evaluation of tendon healing using fibroblast like synoviocytes in rabbits: A biomechanical study.

Tendon never restores the complete biological and mechanical properties after healing. Several techniques are available for tissue-engineered biological augmentation for tendon healing like stem cells. Recently, synovium has been investigated as a source of cells for tissue engineering. In the present study, we investigated potentials of fibroblast like synoviocytes (FLSs) in tendon healing. Sixteen rabbits were divided randomly into control and treatment groups. One rabbit was used as a donor of synovial membrane (synovium). The injury model was unilateral complete transection through the middle one third of deep digital flexor tendon (DDFT). Subsequently, the tendon stumps were sutured with 3/0 nylon. In treatment group, 0.1 mL phosphate-buffered saline (PBS) solution containing 1 × 10(6) nucleated cells of FLSs was injected intratendinously at both tendon stumps just next to incision line. In control group, 0.1 mL PBS without FLSs was used for intratendinous injection. Model animals were euthanized at eight weeks, DDFTs were harvested and prepared for biomechanical study. Results of study showed that, there was no significant differences in biomechanical parameters values between FLSs treated and control groups. In conclusion, intratendinous injection of FLSs did not improve biomechanical properties during eight weeks in rabbit.

Changes of stress oxidative enzymes in rat mammary tissue, blood and milk after experimental mastitis induced by E. coli lipopolysaccharide.

The present study investigated the effects of E. coli lipopolysaccharide (LPS) induced mastitis model in rat on the activity of antioxidant enzyme systems. To achieve this purpose, E. coli LPS were infused into the mammary gland of 12 rats 72 hr postpartum and compared with 12 rats in control group infused intramammary placebo sterile pyrogene - free, physiological saline. The antioxidant activities of the enzymes, superoxide dismutase, glutathione peroxidase, and catalase together with malondialdehyde (MDA) level were assayed in blood serum, milk and mammary tissue. Results obtained showed that, the antioxidant enzyme activities in milk, blood serum and mammary tissue were significantly decreased while the level of MDA, the indicator of lipid peroxidation were significantly increased following intramammary inoculation of LPS compared to the control animals. Histopathological examination also revealed the infiltration of inflammatory cells in mammary tissue and disruption of acinar structure and acinar lumina in mastitic rats. The results indicated that E. coli LPS-induced mastitis could alter antioxidant enzymes and increase lipid peroxidation.

Immunotherapeutic effects of pentoxifylline in type 1 diabetic mice and its role in the response of T-helper lymphocytes.

OBJECTIVES: Pentoxifylline is an immunomodulatory and anti-inflammatory agent and is used in vascular disorders. It has been shown that pentoxifylline inhibits proinflammatory cytokines production. The purpose of this study was to investigate the therapeutic effects of pentoxifylline on the treatment of autoimmune diabetes in mice. MATERIALS AND METHODS: Diabetes was induced by multiple low dose of streptozotocin (MLDS) injection (40 mg/kg/day for 5 consecutive days) in male C57BL/6 mice. After induction of diabetes, mice were treated with pentoxifylline (100 mg/kg/day IP) for 21 days. Blood glucose levels and plasma levels of insulin were measured. Splenocytes were tested for proliferation by MTT test and cytokine production by ELISA. RESULTS: Pentoxifylline treatment prevented hyperglycemia and increased plasma insulin levels in the diabetic mice. Aside from reducing lymphocyte proliferation, pentoxifylline significantly inhibited the production of proinflammatory interleukin 17 (IL-17) as well as interferon gamma (IFN-γ), while increased anti-inflammatory cytokine IL-10 as compared with those in MLDS group (diabetic control group). CONCLUSION: These findings indicate that pentoxifylline may have therapeutic effect against the autoimmune destruction of the pancreatic beta-cells during the development of MLDS-induced type 1 diabetes in mice.