Evaluation the ability of acellular ovine small intestine submucosa to load and release of mineral pitch and its anti-inflammatory effects.

Injury from the severe burn is exacerbated by a persistent inflammatory response. This response is mediated by cytokines and chemokines, which are released from various immune cells, including mast cells. In this study, the ability of the acellular ovine small intestine submucosa (AOSIS) to load and release of Mineral Pitch (MP) was first investigated, and it was found that the preparation of the scaffold by a modified method enables it to load and release water-soluble drugs. Then, 32 male Wistar rats were divided into four groups, a third-degree burn was created, and except for the control group, the others were treated with: AOSIS, WJ-MSCs seeded AOSIS, or AOSIS loaded with WJ-MSCs and MP. Wound sampling on the 5th day after treatment showed that the number of intact and degranulated mast cells in the treatment groups was associated with a decrease compared to the control group. In the last group, this decrease was the largest (and statically significant (p < 0.05)). Also, by measuring the level of inflammatory factors in blood serum, it was found that in the treatment groups compared to the control group, IL-10 was associated with an increase, and TNF-α was associated with a decrease. The changes in inflammatory factors were more significant (p < 0.05) in the last group. So, our results indicate that AOSIS loaded with WJ-MSCs and MP could be used as an innovative tissue-engineered device to control inflammatory condition during burn wound healing.

An Insight on the Role of Altered Function and Expression of Exosomes and MicroRNAs in Female Reproductive Diseases.

Exosomes are small bilayer-lipid membrane vesicles secreted by living cells that are able to transfer regulatory molecules and genetic information from one cell to another. These vesicles are enriched with several nucleic acids including mRNAs, microRNAs (miRNAs), other non-coding RNAs, as well as proteins and lipids. Alterations in the exosomal content and functions are observed in numerous reproductive diseases in both animals and human cases. MicroRNAs, a class of small endogenous RNA molecules, can negatively regulate gene expression at the post-transcription level. Aberrant microRNA expression has been reported in multiple human reproductive diseases such as polycystic ovary syndrome, preeclampsia, uterine leiomyomata, ovarian cancer, endometriosis, and Asherman's syndrome. This study focuses to review recent research on alterations of microRNA expression and the role of exosomes in female reproductive diseases. It has been demonstrated that exosomes may be a potential therapeutic approach in various female reproductive diseases. In addition, changes in expression of microRNAs act as molecular biomarkers for diagnosis of several reproductive diseases in women, and regulation of their expression can potentially reduce infertility.

Using 3D-bioprinting scaffold loaded with adipose-derived stem cells to burns wound healing.

Three dimensional (3D) printing has recently expanded in popularity and has become an effective approach for tissue engineering. Advances in tissue engineering have increased the effectiveness of cell-based therapies. Indeed, the ultimate goal of such treatment is the development of conditions similar to fetal wound regeneration. In this context, technology of 3D printing also allows researchers to more effectively compose multi-material and cell-laden scaffolds with less effort. In this study, we explored a synthetic gel scaffold derived from 3D bioprinter with or without stem cells to accelerate wound healing and skin defects. Adipose-derived stem cells (ADSCs) were isolated and seeded into 3D bioprinter derived-gel scaffold. Morphological and cell adherence properties of 3D scaffold were assessed by hemotoxylin & eosin (H&E) staining and scanning electron microscopy and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide assay. In vivo assessment of the scaffold was done using H&E staining in the full-thickness burn rat model. The experimental groups included; (a) untreated (control), (b) 3D bioprinter derived-gel scaffold (Trial 1), and (c) 3D bioprinter derived-gel scaffold loaded with ADSC (Trial 2). Our results represented 3D bioprinter derived-gel scaffold with or without ADSCs accelerated wound contraction and healing compared to control groups. Epithelization was completed until 21 days after operation in scaffold alone. In scaffold with ADSCs group, epithelization was faster and formed a multi-layered epidermis with the onset of cornification. In conclusion, 3D bioprinter derived-gel scaffold with or without ADSCs has the potential to be used as a wound graft material in skin regenerative medicine.

The useful agent to have an ideal biological scaffold.

Tissue engineering which is applied in regenerative medicine has three basic components: cells, scaffolds and growth factors. This multidisciplinary field can regulate cell behaviors in different conditions using scaffolds and growth factors. Scaffolds perform this regulation with their structural, mechanical, functional and bioinductive properties and growth factors by attaching to and activating their receptors in cells. There are various types of biological extracellular matrix (ECM) and polymeric scaffolds in tissue engineering. Recently, many researchers have turned to using biological ECM rather than polymeric scaffolds because of its safety and growth factors. Therefore, selection the right scaffold with the best properties tailored to clinical use is an ideal way to regulate cell behaviors in order to repair or improve damaged tissue functions in regenerative medicine. In this review we first divided properties of biological scaffold into intrinsic and extrinsic elements and then explain the components of each element. Finally, the types of scaffold storage methods and their advantages and disadvantages are examined.

Study of chondrogenic potential of stem cells in co-culture with chondrons.

OBJECTIVES: Three-dimensional biomimetic scaffolds have widespread applications in biomedical tissue engineering due to similarity of their nanofibrous architecture to native extracellular matrix. Co-culture system has stimulatory effect on chondrogenesis of adult mesenchymal stem cells. This work presents a co-culture strategy using human articular chondrons and adipose-derived stem cells (ASCs) from infrapatellar fat pad (IPFP) for cartilage tissue production. MATERIALS AND METHODS: Isolated stem cells were characterized by flowcytometry. Electrospun and polycaprolactone (PCL) scaffolds (900 nm fiber diameter) was obtained from Bon Yakhteh (Tehran-Iran) and human infrapatellar fat pad-derived stem cells (IPFP-ASCs) were seeded on them. IPFP-ASCs on scaffolds were co-cultured with articular chondrons using transwell. After 21 day, chondrogenic differentiation of stem cell was evaluated by determining the genes expression of collagen2, aggrecan and Indian hedgehog using real-time RT-PCR. RESULTS: Genes expression of collagen2, aggrecan by IPFP-ASCs did not alter significantly in comparison with control group. Howevers, expression of Indian hedgehog decreased significantly compared to control group (P< 0.05). CONCLUSION: These findings indicate that chondrons obtained from osteoarthritic articular cartilage did not stimulate chondrogenic differentiation of IPFP-ASCs in co-culture.

Immunohistochemical (Ki-67) study of endometrial maturation in mice after use of phosphodiesterase type 5 inhibitor.

BACKGROUND: Uterine receptivity for the implantation is a complicated process, that ovarian factors (hormonal), endometrium and embryo simultaneously are involved in this phenomenon. A successful implantation needs appropriate development of the endometrium. Furthermore, embryo must be capable of reacting with the endometrium and producing suitable adhesion molecules. This study aimed to examine one of endometrial maturation indices in mice before implantation, i.e., proliferation of stromal cells. MATERIALS AND METHODS: A total of 40 adult female mice were divided into four groups: Control, gonadotropin, gonadotropin + progesterone, and gonadotropin + sildenafil citrate. The three experimental groups were first injected 7.5 IU of human chorionic gonadotropin (HCG) and then 7.5 IU of human menopausal gonadotropin (HMG). Then, every two female mice were placed in a cage with a male mouse for mating. Two groups were injected 1 mg of progesterone and 3 mg/kg of sildenafil citrate at intervals of 24, 48, and 72 h after injection of HMG. After 96 h, all the mice were killed, and their uterine samples subjected to tissue passage and prepared for analysis. Immunohistochemical method, Ki-67, and stromal mitotic cell count were used in this study. RESULTS: Our observations in all groups showed changes in the luminal epithelium. ANOVA analysis Ki-67-positive stromal cells among all groups were not statistically significant. CONCLUSION: The results showed that administration of HMG and HCG following that of progesterone and sildenafil citrate could change the indices of endometrial maturation, and they were not involved in the phase immediately before implantation in stromal mitotic index.

The effect of pulp and seed extract of Citrullus Colocynthis, as an antidaibetic medicinal herb, on hepatocytes glycogen stores in diabetic rabbits.

BACKGROUND: Medicinal herbs such as Citrullus Colocynthis (C.C) have been used traditionally in the treatment of diabetes mellitus. However therapeutic applications and adverse effects of C.C and its natural variants are not determined well. The current work investigates the effects of pulp and seed extract of C.C on hepatocyte's glycogen stores. MATERIALS AND METHODS: Thirty six male rabbits were divided into six groups (control and diabetic) randomly. Alloxan was used in order to induce diabetes mellitus in animals. Among 5 diabetic groups, one remained as control and the rest received 100 and 200 mg/kg/day of either pulp or seed extract. One month later, animals were sacrificed and their liver specimen fixed in 10% Formalin was stained with periodic acid schiff (PAS) for light microscopic scanning. RESULTS: PAS staining of hepatocytes revealed large amounts of glycogen stores in diabetic animals treated with pulp and seed extracts of C.C, contrary with non-treated diabetic rabbits. Sites of glycogen deposition were also different in animals treated with seed extract (P < 0.0001). No hepatic congestion was seen in treated animals. Dose escalation has no effect on the obtained results. CONCLUSIONS: The anti-diabetic effects of C.C can be explained by its effects on accumulation of glycogen stores in hepatocytes. The importance of varied sites of glycogen deposition by the application of C.C needs to be determined.

Progesterone and ovarian stimulation control endometrial pinopode expression before implantation in mice.

UNLABELLED: Endometrial epithelial pinopodes are considered as markers of endometrial receptivity and seem to be directly involved in the adhesion of the blastocysts to the endometrial surface. The aim of the study was to assess the effects of ovarian stimulation and progesterone injection on pinopode expression, and compare morphological characteristics in the preimplantation stage in mice. Adult female mice (n=30) were divided into three groups: control, superovulated and superovulated-progesterone injected. In experimental groups the mice received 7.5I.U human menopausal gonadotropin (HMG) and then after 48h 7.5I.U human chorionic gonadotropic (HCG) hormone. After that every two females were put with one male in one cage for mating. Superovulated-progesterone group were injected with progesterone (1mg/mouse) in 24, 48, 72h interval after the HMG injection. Animals were sacrificed in 96h after HMG injection, and their uterines (the middle one-third) were prepared for transmission electron microscope studies. That demonstrated that all groups differed from each other. In most of the controls and hyperstimulated-progesterone mice 4 days after HMG injection, long and short microvilli were seen, but they had no developed pinopodes, while in hyperstimulated mice, well developed pinopodes were expressed 4 days after HMG injection. IN CONCLUSION: hyperstimulated mice without progesterone injection may be useful for the studies of pinopodes and implantation.

IGF-1 and PDGF-bb suppress IL-1ß-induced cartilage degradation through down-regulation of NF-κB signaling: involvement of Src/PI-3K/AKT pathway.

OBJECTIVE: Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1ß-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes. METHODS: Primary chondrocytes were treated with IL-1ß to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy. RESULTS: Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1ß-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1ß-induced down-regulation of collagen type II, cartilage specific proteoglycans, ß1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1ß-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1ß signaling. CONCLUSION: The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1ß-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects.

Maternal tamoxifen treatment alters oocyte differentiation in the neonatal mice: inhibition of oocyte development and decreased folliculogenesis.

AIM: Primary germ cells after migration to the developing ovary differentiate to oogonia and oocytes. The formation and number of primordial follicles in early fetal life are determining factors in the fertility state of adult life. Tamoxifen is used both to stimulate ovulation and as an anticancer drug. The aim of the present study was to evaluate the effect of tamoxifen on oocyte and follicular development and differentiation in mice. METHODS: Thirty adult female and 15 adult male mice were used in the present study. The female mice were divided into two groups: control and experimental. Two female mice at their sterous cycle were placed with one male mouse in a cage for mating. The observation of a vaginal plug was considered the 1st day of pregnancy. On the 13th day of pregnancy the mice received 100 microg tamoxifen by i.p. injection. At the end of pregnancy 2-, 3-, 6- and 7-day-old newborns were killed and their ovaries were fixed and prepared for light and electron microscopic studies. The number of follicular nests and diameter of primordial and primary follicles were determined using Motic software (Motic Incorporation Ltd, Canada) and compared with control values using t-test. RESULTS: Microscopy and morphometry showed that oocyte nests are formed on the 2nd and 3rd days and follicles are distinguished on the 6th and 7th days. Morphometric studies revealed that the number and diameters of oocyte nests were significantly (P < 0.001) reduced in the experimental groups compared to the control group. However, the numbers and diameters of primordial and primary follicles in experiential and controls were not significantly different. Electron microscopic studies revealed that in the control group, oocytes were separated from each other and were at primordial follicle stage. However, in the experimental group, the oocytes were in clusters as oocyte nests. CONCLUSION: The results indicate that tamoxifen suppresses follicular differentiation at early stages but does not affect the development of already differentiated follicles.

Dietary L-arginine and cutaneous wound healing.

Skin wound healing has been the subject of extensive studies and various drugs have been used in an attempt to improve wound healing. There are conflicting data regarding the effects of L-arginine, the substrate of nitric oxide, on wound healing. We examined the 1-week rate of cutaneous wound healing and collagen deposition in three groups of rats who received a (1) L-arginine (2% in drinking water)-supplemented diet from three days before until the seventh day following injury (Group 1), (2) L-arginine-supplemented diet for three days before injury (Group 2), and (3) a standard diet without L-arginine supplementation (Group 3). The wound length and width were measured each day and then the open wound area and cumulative percentage of open wound area reduction were calculated. Wound biopsy samples were examined with Trichrome-Masson stain in a subgroup of animals. Results showed that Group 1 rats had a significantly lower cumulative percentage of open wound area reduction on day 7 compared to other two groups (Mann-Whitney U test, P < 0.05). Relatively higher degrees of wound collagen deposit (day 7) were noted in groups 2 and 3. It may be concluded that L-arginine (2% in water) administered three days before until the seventh day following skin wound induction may diminish the rate of skin wound healing and collagen deposition.

Morphometric study of GnRH analog/HMG/HCG effects on ultrastructure of human endometrial epithelium in early and mid-luteal phase.

AIM: The aim of the present study was to investigate the effects of long protocol ovulation induction on the ultrastructure of the human endometrial epithelium on days luteinizing hormone (LH) + 4 and LH+ 6 of the menstrual cycle. METHODS: Endometrial biopsies were obtained on days LH+ 4 (n = 9) and LH+ 6 (n = 10) from infertile women who were under standard long protocol with luteal phase supplementation with IM (intramuscular) progesterone, but where the embryo had not been formed or transferred, due to the male factor problem. Biopsies were also taken on days LH+ 4 (n = 5) and LH+ 6 (n = 5) from fertile women who had not received ovulation induction drugs as control groups. After preparation and taking light and electron micrographs from samples, qualitative and quantative evaluations (morphological and morphometric) were accomplished and the data was compared using the unpaired student t-test. RESULTS: Qualitative results showed the presence of the nuclear channel system, vacuoles of glycogen and giant mitochondria in all of the samples. Qualitative analysis showed that the volume fraction (Vv) of the euchromatin to the nucleus, the rough endoplasmic reticulum and the mitochondria to the cell, were not statistically different (P > 0.05) in samples taken on LH+ 4 in both control and test groups. The Vv of these features, however, to the cell in the test group was significantly (P < 0.05) higher than those in the control taken on LH+ 6. CONCLUSIONS: These results suggest that long protocol ovulation induction with luteal phase support with progesterone alter the normal development of the human endometrium in the mid luteal phase and could decrease the implantation success rate.

Fluoxetine enhances cutaneous wound healing in chronically stressed Wistar rats.

OBJECTIVES: To evaluate the histologic and clinical effects of fluoxetine administration on wound healing in chronically stressed and nonstressed Wistar rats. STUDY DESIGN: Full-thickness incisional wounds were created on the lower back of 72 female Wistar rats. Animals were divided into 2 stress and nonstress groups according to application of stress regimen and 3 subdivisions based on placebo, acute, or chronic administration of fluoxetine. Wound length, width, and linear healing rate based on wound area were measured for 2 weeks postwounding. Biopsies of 3 rats from each group were taken at days 1, 4, 7, and 14 to perform histomorphometric measurements by light microscopy. Analysis of covariance and analysis of variance were used to analyze wound length and other variables, respectively. RESULTS: Fluoxetine treatment significantly reduced mean wound length and healing period (P<.01). Although stress decreased the linear healing rate by 48%, fluoxetine treatment increased it by 68% and 31% in stressed and nonstressed rats, respectively. Stress significantly diminished infiltration of neutrophils and monocytes (P<.01), disrupted spatial organization of fibroblasts, and delayed neovascularization. Fluoxetine precluded these effects successfully. CONCLUSION: Fluoxetine significantly improves healing of cutaneous wounds in stressed and, to a lesser extent, in nonstressed animals.

Ultrastructural alterations and occurrence of apoptosis in developing follicles exposed to low frequency electromagnetic field in rat ovary.

Humans are continuously exposed to harmful environmental factors, including Electromagnetic Fields (EMF) produced by home appliances, diagnostic tools and industrial instruments. The present study investigates morphologically the effects of EMF on ovarian follicles. Female Wistar rats were exposed to 3 mili Tesla EMF for 4 h day(-1) for 4 months, the ovaries from both experimental and control groups were processed for Transmission Electron Microscopy (TEM) and TUNEL reaction technique. TEM revealed that in the oocytes from experimental group the nuclei were shrunken and zona pellucida appeared narrower than those in the control group. The number of microvilli were decreased significantly and in their cytoplasm there were several lipid droplets and organels were dispersed. Signs of apoptosis such as condensed nuclei, chromatin margination and nuclear membrane dilatation were common in granulosa and corona radiata cells from the EMF-exposed rats than those from the control group. Primary morphological changes in the granulosa cells from this group were retraction of granulosa cells, loss of microvilli and condensation or loss of mitochondrial cristae. The morphological changes in oocytes seem to indicate the cytotoxic effect of EMF and the changes occurred in granulosa cells coincide with initiation of apoptosis in granulosa cells. Present results suggest that EMF exposure may interfere with normal process of folliculogenesis.