Thymoquinone loading into hydroxyapatite/alginate scaffolds accelerated the osteogenic differentiation of the mesenchymal stem cells.

BACKGROUND: Phytochemical agents such as thymoquinone (TQ) have osteogenic property. This study aimed to investigate the synergic impact of TQ and hydroxyapatite on mesenchymal stem cell differentiation. Alginate was also used as drug vehicle. METHODS: HA scaffolds were fabricated by casting into polyurethane foam and sintering at 800 °C, and then, 1250 °C and impregnated by TQ containing alginate. The adipose-derived stem cells were aliquoted into 4 groups: control, osteogenic induced-, TQ and osteogenic induced- and TQ-treated cultures. Adipose derived-mesenchymal stem cells were mixed with alginate and loaded into the scaffolds RESULTS: The results showed that impregnation of HA scaffold with alginate decelerated the degradation rate and reinforced the mechanical strength. TQ loading in alginate/HA had no significant influence on physical and mechanical properties. Real-time RT-PCR showed significant elevation in collagen, osteopontin, and osteocalcin expression at early phase of differentiation. TQ also led to an increase in alkaline phosphatase activity. At long term, TQ administration had no impact on calcium deposition and proliferation rate as well as bone-marker expression. CONCLUSION: TQ accelerates the differentiation of the stem cells into the osteoblasts, without changing the physical and mechanical properties of the scaffolds. TQ also showed a synergic influence on differentiation potential of mesenchymal stem cells.

Synergic effects of decellularized bone matrix, hydroxyapatite, and extracellular vesicles on repairing of the rabbit mandibular bone defect model.

BACKGROUND: Extracellular vesicles (ECV) and bone extracellular matrix (ECM) have beneficial effects on the treatment of some pathological conditions. The purpose of this study was to find the synergic effects of decellularized bone (DB) ECM and ECVs on the repair of rabbit. METHODS: The quality of decellularized sheep bones was confirmed by H&E, Hoechst, DNA quantification, immunohistochemistry, histochemical staining, and scanning electron microscopy (SEM). Osteoblast-derived ECVs were evaluated by internalization test, Transmission electron microscopy, Dynamic light scattering, and flow cytometry for CD9, CD63, CD81 markers. The hydrogel containing DB and hydroxyapatite (HA) with or without ECVs was evaluated for osteoblast functions and bone repair both in vitro and in vivo. RESULTS: The data indicated ECM preservation after decellularization as well as cell depletion. In vitro assessments revealed that mineralization and alkaline phosphatase activity did not improve after treatment of MG63 cells by ECVs, while in vivo morphomatrical estimations showed synergic effects of ECVs and DB + HA hydrogels on increasing the number of bone-specific cells and vessel and bone area compared to the control, DB + HA and ECV-treated groups. CONCLUSIONS: The DB enriched with ECVs can be an ideal scaffold for bone tissue engineering and may provide a suitable niche for bone cell migration and differentiation.

Lectin Profile Variation in Mesenchymal Stem Cells Derived from Different Sources.

Human mesenchymal stem cells (MSCs), a promising source of stem cells for regenerative medicine, have different morphological and functional characteristics. Carbohydrate moieties on the cell surface play an important role, including cell-cell interaction and cell recognition. The objective of this study was to determine possible differences in glycoconjugate distribution patterns of MSCs derived from various sources. MSCs were isolated from adipose tissue, bone marrow, Wharton's jelly, and cord blood. Then, they were stained with FITC-conjugated wheat germ agglutinin (WGA), peanut agglutinin (PNA), concanavalin A (ConA), Ulex europaeus (UEA), Dolichos biflorus (DBA), and Atto-488 conjugated Phytolacca americana (PWM) lectins. The intensity of the reactions was scored using ImageJ software. Flow cytometry was performed to detect the expression of the endothelial marker CD144. The obtained data were analyzed by ANOVA and LSD. Cord blood-derived MSCs showed the most significant staining intensities with all lectins. All MSCs were also moderately stained with PNA. Bone marrow-derived MSCs failed to react with UEA, DBA, and ConA. Wharton's jelly-derived MSCs could also not be stained with ConA. Cord blood-derived MSCs contained 2 subpopulations: osteoclast- and fibroblast-like cells. Both lectin staining intensity and distribution pattern were different in these 2 cell types; therefore, the central part of osteoclast-like cells stained more intensive with PNA and PWM, while that part in fibroblast-like cells stained more intensive with ConA. None of them expressed CD144. The glycoconjugate content of MSCs derived from various sources is different.

Effects of platelet-rich plasma on liver regeneration in CCl4-induced hepatotoxicity model.

Numerous bioactive growth factors and cytokines in platelet-rich plasma (PRP) have recently made it an attractive biomaterial for therapeutic purposes. These growth factors have the potential to regenerate the injured tissues. The aim of this study was to investigate the therapeutic effects of PRP in hepatotoxic animal model. Hepatotoxicity was induced in rats by oral administration of 4 mL/kg/week of CCl4 diluted 1:1 in corn oil for 10 weeks. To confirm the hepatotoxicity, 24 h after the last CCl4 administration, blood samples were collected via cardiac puncture to assess the serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, total protein, and total bilirubin. Twenty-four hours after blood collection, the experimental animals received a single injection of PRP (1 mL) via the anterior mesenteric vein. One week later, all biochemical tests were performed again, and the rats were scarified and their livers were removed, prepared histologically, and stained. The stereological analyses were performed to evaluate the effects of PRP on histopathological features of CCl4-treated livers. The results were compared statistically with the corresponding control and CCl4+normal saline (NS)-treated animals. A significant decrease in the number and volume of hepatocytes (p = 0.01), and also a reduction in the volume of sinusoids (p = 0.001) and connective tissue (p = 0.04), were observed in the PRP-treated animals compared with the CCl4+NS-treated ones. Our findings demonstrated that application of PRP had beneficial effects on CCl4-induced fibrosis; however, it had detrimental effects on the total number of hepatocytes and the volume of hepatocytes and sinusoidal spaces.

Comparison of the Expression of Hepatic Genes by Human Wharton's Jelly Mesenchymal Stem Cells Cultured in 2D and 3D Collagen Culture Systems.

BACKGROUND: Human Wharton's jelly mesenchymal stem cells (HWJMSCs) express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naïve HWJMSCs. METHODS: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes. RESULTS: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 (HNF4) was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold. CONCLUSION: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other.

Wharton's Jelly-derived Mesenchymal Stem Cells can Differentiate into Hepatocyte-like Cells by HepG2 Cell Line Extract.

BACKGROUND: Wharton's jelly is an unlimited source of stem cells that can be used in cell therapy and tissue engineering without any ethical concern. It has been revealed the cell-free extract could be effective to induce cell differentiation. The objective of this study was to induce Wharton's jelly-derived mesenchymal stem cells (MSCs) into hepatocyte-like cells by premeabilization of the cells in the presence of HepG2 cell line extract. METHODS: MSCs were isolated from the umbilical cord, CD marker profile and their differentiation potential into adipogenic and osteogenic lineages were determined. The cells were then, permeabilized by streptolysin O in the presence of HepG cell extract. The treated cells were cultured for 17 days. The cell phenotype was evaluated and the hepatocyte specific markers were detected by immunofluorescence and immunocytochemistry. The Periodic Acid Schiff (PAS) reaction and the cellular uptake of indocyanine green were performed to evaluate the functional behavior of the differentiated cells. RESULTS: The phenotype of extract-treated MSCs changed into a round or polygonal cells with few short processes and they could express high level of albumin, cytokeratin 18 and 19. The MSCs also could store glycogen and uptake and release indocyanine green. CONCLUSION: We demonstrated for the first time that Wharton's jelly-derived MSCs could differentiate into hepatocyte-like cells by premeabilization of them in the presence of HepG2 cell extract. This study suggests a feasible method to differentiate MSCs into functional hepatocyte-like cells.

Cardiomyocyte marker expression in mouse embryonic fibroblasts by cell-free cardiomyocyte extract and epigenetic manipulation.

BACKGROUND: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. METHODS: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. RESULTS: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. CONCLUSION: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function.

Optimizing artificial meniscus by mechanical stimulation of the chondrocyte-laden acellular meniscus using ad hoc bioreactor.

BACKGROUND: Tissue engineering focuses on reconstructing the damaged meniscus by mimicking the native meniscus. The application of mechanical loading on chondrocyte-laden decellularized whole meniscus is providing the natural microenvironment. The goal of this study was to evaluate the effects of dynamic compression and shear load on chondrocyte-laden decellularized meniscus. MATERIAL AND METHODS: The fresh samples of rabbit menisci were decellularized, and the DNA removal was confirmed by histological assessments and DNA quantification. The biocompatibility, degradation and hydration rate of decellularized menisci were evaluated. The decellularized meniscus was injected at a density of 1 × 105 chondrocyte per scaffold and was subjected to 3 cycles of dynamic compression and shear stimuli (1 h of 5% strain, ± 25°shear at 1 Hz followed by 1 h rest) every other day for 2 weeks using an ad hoc bioreactor. Cytotoxicity, GAG content, ultrastructure, gene expression and mechanical properties were examined in dynamic and static condition and compared to decellularized and intact menisci. RESULTS: Mechanical stimulation supported cell viability and increased glycosaminoglycan (GAG) accumulation. The expression of collagen-I (COL-I, 10.7-folds), COL-II (6.4-folds), aggrecan (AGG, 3.2-folds), and matrix metalloproteinase (MMP3, 2.3-folds) was upregulated compared to the static conditions. Furthermore, more aligned fibers and enhanced tensile strength were observed in the meniscus treated in dynamic condition with no sign of mineralization. CONCLUSION: Compress and shear stimulation mimics the loads on the joint during walking and be able to improve cell function and ultrastructure of engineered tissue to recreate a functional artificial meniscus.

Prednisolone and mesenchymal stem cell preloading protect liver cell migration and mitigate extracellular matrix modification in transplanted decellularized rat liver.

INTRODUCTION: Regenerative medicine provides promising approaches for treating chronic liver diseases. Previous studies indicate that decellularized liver architecture is damaged by invading non-hepatic inflammatory cells. This study aimed to use anti-inflammatory and regenerative potency of bone marrow-derived mesenchymal stem cells (BM-MSC) and prednisolone for reducing fibrosis and balancing inflammatory cell migration into the decellularized liver scaffold. MATERIAL AND METHOD: The liver was decellularized by perfusing Sodium Lauryl Ether Sulfate (SLES), and nuclei depletion and extracellular matrix (ECM) retention were confirmed by DNA quantification, histochemical, and immunohistochemical assessments. Scaffolds were loaded with BM-MSCs, prednisolone, or a combination of both, implanted at the anatomical place in the rat partial hepatectomized and followed up for 2 and 4 weeks. RESULTS: Labeled-MSCs were traced in the transplanted scaffolds; however, they did not migrate into the intact liver. Immunohistochemistry showed that the hepatoblasts, cholangiocytes, stellate, and oval cells invaded into all the scaffolds. Bile ducts were more abundant in the border of the scaffolds and intact liver. Stereological assessments showed a significant reduction in the number of lymphocytes and neutrophils in prednisolone-loaded scaffolds. The regeneration process and angiogenesis were significantly higher in the group treated with cell/prednisolone-loaded bioscaffolds. Collagen fibers were significantly reduced in the scaffolds pre-treated with cell/prednisolone, prednisolone, or BM-MSCs, compared to the control group. CONCLUSION: Loading prednisolone into the scaffolds can be a worthy approach to restrict inflammation after transplantation. Although pre-loading of the scaffolds with a combination of cells/prednisolone could not alleviate inflammation, it played an important role in regeneration and angiogenesis.

In Situ Casting of Platelet Rich Plasma/SiO2/Alginate for Bone Tissue Engineering Application in Rabbit Mandible Defect Model.

Statement of the Problem: The administration of both platelet rich plasma (PRP) and silicon dioxide (SiO2) to the bone defects accelerates bone repair and regeneration. Appli-cation of both of them may show synergistic regenerative effects. Purpose: Our objective was to evaluate the possible synergistic osteogenic effects of PRP and SiO2 by injecting them using an ad hoc device. Materials and Method: In this experimental study, PRP/SiO2 scaffolds were fabricated by in situ casting method with the help of CaCl2 as the gelation factor and alginate as the stroma; and then, the biodegradability and spatial arrangement were assessed. The injecta-ble scaffold was introduced into the 40 rabbit mandibular defects by an ad hoc two-channel injecting device. Five defects received PRP/SiO2/alginate as the treatment; the other sets of defects were treated by PRP/alginate, SiO2/alginate, and the last five defects served as the control groups by getting only alginate injections. The osteogenicity of the scaffolds was evaluated by radiological and histological procedures; they were then compared with each other. Analysis of variance and least significant difference tests were used to analyze the data. Results: The SiO2-treated group showed a significant higher bone area compared to PRP/ SiO2-treated groups on day 40 (p= 0.013). The number of osteocytes was higher in SiO2-treated than the control groups on both 20 and 40 days (p= 0.032 and 0.022, respectively). The number of osteoclast was also higher in SiO2-treated than PRP-treated group (p= 0.028). In addition, the cells of this group had just started to create Haversian systems in newly formed bone tissues. Conclusion: Silica demonstrated a superior osteogenic activity over PRP in both short and long term periods. Evidently, they showed no synergistic regenerative effects. Our ad hoc device was efficiently capable of inserting the scaffolds into the injured sites with no diffi-culties or complications.

Comparative assessment of the efficiency of various decellularization agents for bone tissue engineering.

Bone regeneration can be possible through grafts or engineered bone replacement when bone defects are larger than the critical size. Decellularized bone extracellular matrix (ECM) is an alternative that is able to accelerate tissue regeneration, while decellularization protocols influence engineered bone quality. The objective of this study was to compare the quality of decellularized bone produced through different methods. Four decellularization methods were employed using (a) sodium lauryl ether sulfate (SLES), (b) sodium dodecyl sulfate (SDS) 0.5%, (c) SDS 1% and (d) trypsin/EDTA. All samples were then washed in triton X-100. DNA quantification, hematoxylin and eosin, and Hoechst staining showed that although DNA was depleted in all scaffolds, treatment with SLES led to a significantly lower DNA content. Glycosaminoglycan quantification, Raman confocal microscopy, alcian blue and PAS staining exhibited higher carbohydrate retention in the scaffolds treated with SLES and SDS 0.5%. Raman spectra, scanning electron microscopy and trichrom Masson staining showed more collagen content in SLES and SDS-treated scaffolds compared to trypsin/EDTA-treated scaffolds. Therefore, although trypsin/EDTA could efficiently decellularize the scaffolds, it washed out the ECM contents. Also, both MTT and attachment tests showed a significantly higher cell viability in SLES-treated scaffolds. Raman spectra revealed that while the first washing procedure did not remove SLES traces in the scaffolds, excessive washing reduced ECM contents. In conclusion, SLES and, to a lesser degree, SDS 0.5% protocols could efficiently preserve ultrastructure and ECM constituents of decellularized bone tissue and can thus be suggested as nontoxic and safe protocols for bone regeneration.

Decellularized human ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated protocol, as a natural three-dimensional scaffold for construction of bioengineered ovaries.

BACKGROUND: The increasing number of patients with ovarian insufficiency due to autoimmune disorders, genetic predisposition, or iatrogenic effects of treatment such as cancer therapies necessitates an urgent measure to find a safe and transplantable alternative ovary. A bioengineered ovary is one of the strategies on which the researchers have recently been working. An engineered ovary should be able to mimic the natural ovary aspects. Recent studies suggest that the decellularized organ-specific extracellular matrix-based scaffolds can serve as a native niche to bioengineering artificial organs. Therefore, we established a human decellularized ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated process, as an optimized protocol. METHODS: The human ovary samples were decellularized with 1% SLES for 48 h followed by DNase I in PBS for 24 h, and then thoroughly rinsed in PBS to remove the cell remnants and chemical reagents. Efficient cell removal was confirmed by DNA content analysis, hematoxylin and eosin, and Hoechst staining. Preservation assessment of the extracellular matrix structures was performed by immunohistochemistry, histological staining, and scanning electron microscopy. An MTT test was done to assess the in vitro scaffold's cytocompatibility, and finally in vivo studies were performed to evaluate the biocompatibility, bioactivity, and secretion functions of the ovarian grafts made of primary ovarian cells (POCs) on the decellularized scaffolds. RESULTS: Evidence provided by SEM, histochemical, and immunohistochemical analyses showed that the ovarian extracellular matrix was preserved after decellularization. Moreover, MTT test indicated the suitable cytocompatibility of the scaffolds. The in vivo assessment showed that the POCs kept their viability and bioactivity, and reconstructed the primordial or primary follicle-like structures within the scaffolds after transplantation. Immunostaining characterized somatic cells that were capable of expressing steroid hormone receptors; also, as a marker of granulosa cell, inhibin-α immunostaining demonstrated these cells within the grafts. Additionally, hormone assessment showed that serum estradiol and progesterone levels were significantly higher in ovariectomized rats with ovarian cells-seeded grafts than those with or without decellularized scaffold grafts. CONCLUSIONS: A human ovary-specific scaffold based on a SLES-decellularized protocol as a biomimicry of the natural ovarian niche can be an ideal scaffold used to reconstruct the ovary.

Fetal microchimerism in mouse caerulein-induced pancreatitis model.

OBJECTIVES: Fetal microchimerism is the persistence of allogeneic cell population that transfer from the fetus to the mother. The aim of this study was to evaluate the presence of fetal microchimerism in the pancreas of the mouse with acute pancreatitis (AP). MATERIALS AND METHODS: In this experimental study, female wild-type mice were mated with male EGFP+. AP model was obtained by injection of caerulein two days after delivery. Sixty mice were divided into 3 groups: the virgin pancreatitis-induced animals, pregnant pancreatitis-induced animals mated with transgenic EGFP mice, and pregnant sham animals. To prove pancreatitis induction, the blood amylase and lipase were assessed; and pancreas was removed from a subpopulation of each group for histopathological examinations after 6 hr. The remaining mice were kept for 3 weeks and histopathological exanimation, immunohistochemistry, and PCR were performed. RESULTS: EGFP+ cells were found in acini and around the blood vessels in the pancreas of pregnant pancreatitis-induced animals. They differentiated to acinar, adipocyte-like, and mesenchymal-like cells. PCR showed that 20% of the pregnant pancreatitis-induced animals were EGFP+. The histopathological study showed improvement in pancreatitis scores in the mice with history of pregnancy. CONCLUSION: It seems that pregnancy has a beneficial impact on caerulein-induced pancreatitis and improves the pancreatitis score in mouse.

Characterization, recellularization, and transplantation of rat decellularized testis scaffold with bone marrow-derived mesenchymal stem cells.

BACKGROUND: Regenerative medicine potentially offers the opportunity for curing male infertility. Native extracellular matrix (ECM) creates a reconstruction platform to replace the organs. In this study, we aimed to evaluate the efficiency of the testis decellularized scaffold as a proper niche for stem cell differentiation toward testis-specific cell lineages. METHODS: Rats' testes were decellularized by freeze-thaw cycle followed by immersion in deionized distilled water for 2 h, perfused with 1% Triton X-100 through ductus deferens for 4 h, 1% SDS for 48 h and 1% DNase for 2 h. The decellularized samples were prepared for further in vitro and in vivo analyses. RESULT: Histochemical and immunohistochemistry studies revealed that ECM components such as Glycosaminoglycans (GAGs), neutral carbohydrate, elastic fibers, collagen I & IV, laminin, and fibronectin were well preserved, and the cells were completely removed after decellularization. Scanning electron microscopy (SEM) showed that 3D ultrastructure of the testis remained intact. In vivo and in vitro studies point out that decellularized scaffold was non-toxic and performed a good platform for cell division. In vivo implant of the scaffolds with or without mesenchymal stem cells (MSCs) showed that appropriate positions for transplantation were the mesentery and liver and the scaffolds could induce donor-loaded MSCs or host migrating cells to differentiate to the cells with phenotype of the sertoli- and leydig-like cells. The scaffolds also provide a good niche for migrating DAZL-positive cells; however, they could not differentiate into post meiotic-cell lineages. CONCLUSION: The decellularized testis can be considered as a promising vehicle to support cell transplantation and may provide an appropriate niche for testicular cell differentiation.

Hepatogenic Differentiation Capacity of Human Wharton's Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract.

BACKGROUND: Human Wharton's jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development. MATERIALS AND METHODS: HWJMSCs were extracted from human Wharton's jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/ collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry. RESULTS: According to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19. CONCLUSIONS: Co-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.

Fabrication and characterization of platelet-rich plasma scaffolds for tissue engineering applications.

Platelet-Rich Plasma (PRP), as a rich source of growth factor, can form a fibrin gel that recapitulates the extracellular matrix of the tissues. The aim of this study was to evaluate the effects of different concentrations of CaCl2 on the PRP scaffold structure which in turn could change the cell's behavior. PRP was mixed with 2.5, 5 and 10% (w/v) CaCl2. Then, the tensile strength, biodegradability and water content of the scaffolds were evaluated. We also performed immunostaining for assessment of the actin stress fiber orientation and SEM for detecting the cell phenotype and physical properties of the fibers. Cell viability, attachment and migration were also evaluated. The highest cell attachment and short term proliferation rate was observed on the scaffolds with 2.5% CaCl2. The cells cultured on the scaffold with higher CaCl2 concentration had fusiform phenotype with few cell processes and parallel arrangement of stress fibers while those cultured on the other scaffolds were fibroblast-like with more processes and net-like stress fibers. The scaffolds with 10% CaCl2 demonstrated the highest osmolarity (358.75±4.99mOsmole), fiber thickness (302.1±54.3nm), pore size (332.1±118.9nm2) and the longest clotting time (12.2±0.776min) compared with the other scaffolds. Water content, branching angle, porosity, orientation and tensile strength did not change by gelation with different CaCl2 concentrations. In conclusion, the cell shape, viability and proliferation were modified by culturing on the PRP scaffolds prepared with various concentrations of CaCl2, and as a result, the scaffolds showed different physical and biological properties.

The effect of amniotic membrane extract on umbilical cord blood mesenchymal stem cell expansion: is there any need to save the amniotic membrane besides the umbilical cord blood?

OBJECTIVES: Umbilical cord blood is a good source of the mesenchymal stem cells that can be banked, expanded and used in regenerative medicine. The objective of this study was to test whether amniotic membrane extract, as a rich source of growth factors such as basic-fibroblast growth factor, can promote the proliferation potential of the umbilical cord mesenchymal stem cells. MATERIALS AND METHODS: The study design was interventional. Umbilical cord mesenchymal stem cells were isolated from voluntary healthy infants from hospitals in Shiraz, Iran, cultured in the presence of basic-fibroblast growth factor and amniotic membrane extracts (from pooled - samples), and compared with control cultures. Proliferation assay was performed and duplication number and time were calculated. The expression of stem cell's specific markers and the differentiation capacity toward osteogenic and adipogenic lineages were evaluated. RESULTS: Amniotic membrane extract led to a significant increase in the proliferation rate and duplication number and a decrease in the duplication time without any change in the cell morphology. Both amniotic membrane extract and basic-fibroblast growth factor altered the expressing of CD44 and CD105 in cell population. Treating basic-fibroblast growth factor but not the amniotic membrane extract favored the differentiation potential of the stem cells toward osteogenic lineage. CONCLUSION: The amniotic membrane extract administration accelerated cell proliferation and modified the CD marker characteristics which may be due to the induction of differentiation toward a specific lineage. Amniotic membrane extract may enhance the proliferation rate and duplication number of the stem cell through changing the duplication time.

Does metformin improve in vitro maturation and ultrastructure of oocytes retrieved from estradiol valerate polycystic ovary syndrome-induced rats.

BACKGROUND: Metformin decreases polycystic ovary syndrome (PCOS) symptoms, induces ovulation, and may improve developmental competence of in vitro oocyte maturation. This study was designed to define the effects of metformin on the characteristics of in vitro oocyte maturation in estradiol valerate (EV) PCOS-induced rats. METHODS: Forty-five adult female Sprague-Dawley rats were randomly divided into control; sham and PCOS-induced (treated by a single dose of estradiol valerate, 4 mg/rat, IM) groups. The body weight was measured weekly for 12 weeks. At the end of week 12, the serum levels of testosterone, estrogen, progesterone, LH, and FSH and blood glucose of all the rats were measured. About 380 cumulus oocyte complexes (control, 125; sham, 122; PCOS-induced rats, 133) were incubated in Ham's F10 in the absence and/or presence of metformin (M 5(-10)) for 12, 24, 36, and 48 h. The cumulus cells expansion and nuclear and cytoplasmic maturation of the oocytes was evaluated using 1 % aceto-orcein staining, and transmission electron microscopy (TEM). RESULTS: No significant differences were observed in the body weight of the rats. The serum level of testosterone was reduced, and progesterone and LH were significantly increased in the PCOS-induced rats (p < 0.05). However, no significant differences were observed in the serum levels of estrogen and FSH among the groups. Blood glucose level was higher in the PCOS-induced rats than control, (p < 0.01). The expansion of cumulus cells was observed in the metformin-treated oocytes. The oocytes retrieved from PCOS-induced rats show a stage of meiotic division (GVBD, MI, A-T, and MII) in 57.12 % of metformin-untreated and fairly significantly increased to 64.28 % in metformin-treated oocytes, (p < 0.05), but no differences were observed in the MII stage within groups. The redistribution of some cytoplasmic organelles throughout the ooplasm, particularly the peripheral cortical granules, was defined in the metformin-treated oocytes. CONCLUSIONS: Single dose of EV can creates a reversible PCO adult rat model. Metformin enhances the COCs to initiate meiotic resumption at the first 6 h of IVM. In our study the metformin inability to show all aspects of in vitro oocyte maturation and may be resulted from deficiency of EV to induce PCOS.

An in vitro model for hepatocyte-like cell differentiation from Wharton's jelly derived-mesenchymal stem cells by cell-base aggregates.

AIM: The present study investigated the differentiation potential of human Umbilical Cord Mesenchymal Stem Cells (UCMSCs) into hepatic lineage through embryonic body-like aggregate formation in the presence of IGF-1. BACKGROUND: Cells derived from Wharton's jelly have been reported to display a wide multilineage differentiation potential, showing some similarities to both embryonic (ESC) and mesenchymal stem cells (MSCs). PATIENTS AND METHODS: Human MSCs isolated from the umbilical cord were plated in 20 µL micro drops. A two-step differentiation protocol was used and the cell aggregates were exposed to the media supplemented with IGF, HGF, oncostatin M, and dexamethasone for 21 days. Immunoperoxidase and immuno-fluorescence were performed for cyrokeratins 18, 19 and albumin. Functional assays were done by periodic acid Schiff (PAS) and indocyanine green. RESULTS: The expression of cytokeratin 19 was shown to be higher in the cells derived from 3D spheroids compared to those cultured in conventional protocol. They showed a polygonal shape after being exposed to hepatogenic media. Immunostaining demonstrated the expression of cytokeratin-18, 19 and albumin by the differentiated cells. Besides, PAS staining revealed glycogen storage in differentiated cells. Also, a greater number of large size differentiated cells were found at the periphery of the expanded cell aggregates. CONCLUSION: We established a protocol for UCMSC differentiation into hepatocytes and these cells were morphologically and functionally similar to hepatocytes. Thus, hepatocyte differentiation may be facilitated by the UCMSCs aggregate formation before administration of the differentiation protocols.

The influence of fibroblast growth factor 4 on hepatogenic capacity of Wharton's jelly mesenchymal stromal cells.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) derived from human umbilical cords could be an appropriate candidate for hepatocyte replacement therapy. Improvement of the efficiency of the cell expression of liver specific genes can be considered in finding new transplantation resources. The present study aimed to differentiate WJ-MSCs toward hepatocyte-like cells on collagen film in the presence of hepatogenic factors, including fibroblast growth factor 4 (FGF4), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). MSCs derived from Wharton's jelly explants were characterized by flow cytometry. Then, the cells were cultured in the presence of hepatogenic media with or without FGF4 on 2D collagen films for 21 days. The expression of liver-specific genes was evaluated by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. The functional assays were performed by Periodic Acid-Schiff (PAS) staining and Indocyanin Green (ICG) uptake. The cultures pre-exposed to FGF4 expressed higher levels of endodermal markers, such as albumin, compared to the control cultures. Also, cytokeratin 18 expression was significantly increased in FGF4-treated cells. However, the expression level of other liver-specific markers was not influenced by exposure to hepatogenic media with or without FGF4. In conclusion, it was demonstrated that FGF4 could induce the differentiation of WJ-MSCs toward endoderm. Despite the morphological changes and increase in PAS reaction, WJ-MSCs could not differentiate into hepatocytes by hepatogenic media consisting of IGF-1.