Anti-inflammatory and healing effect of the polysaccharidic extract of Opuntia ficus-indica cladodes in cutaneous excisional wounds in rats.

This study aimed to investigate the anti-inflammatory and wound healing effects of the polysaccharide extract from Opuntia ficus-indica cladodes (TPL-Ofi) using a rat cutaneous wound model. After anaesthesia, four 7-mm-diameter dorsal wounds per animal (n = 6/group for each experimental day of evaluation) were created in female Wistar rats using a surgical punch. The animals were treated topically twice daily with TPL-Ofi (0.01-1%; treated group) or sterile saline (control group) for a period of 21 days. Ulcerated tissue was collected for analysis of histological parameters (inflammation score, number of polymorphonuclear, mononuclear, fibroblast/myofibroblasts and blood vessels), immunohistochemical (fibroblast growth factor 2 [FGF-2]) and oxidative stress markers (myeloperoxidase [MPO] and glutathione [GSH]). After 21 days of treatment, body weight, net organ weight and plasma biochemical levels were measured. TPL-Ofi, containing a total carbohydrate content of 65.5% and uronic acid at 2.8%, reduced oedema on the second day and increased the nociceptive threshold on the second and third days. TPL-Ofi reduced mononuclear infiltrate on the second and MPO activity on the fifth day. TPL-Ofi increased GSH levels on the second day, as well as fibroblast/myofibroblasts counts, neoangiogenesis and FGF-2 levels on the fifth and seventh days. No changes were observed in body weight, net organ weight or toxicology assessment. Topical application of TPL-Ofi exhibited anti-inflammatory and antinociceptive effects, ultimately improving wound healing in cutaneous wounds.

Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor ß (TGFß), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

Feeding rumen-protected lysine prepartum alters placental metabolism at a transcriptional level.

Rumen-protected Lys (RPL) fed to Holstein cows prepartum resulted in a greater intake and improved health of their calves during the first 6 wk of life. However, whether increased supply of Lys in late gestation can influence placental tissue and, if so, which pathways are affected remain to be investigated. Therefore, we hypothesize that feeding RPL during late gestation could modulate placental metabolism, allowing for improved passage of nutrients to the fetus and thus influencing the offspring development. Therefore, we aimed to determine the effects of feeding RPL (AjiPro-L Generation 3, Ajinomoto Health and Nutrition North America) prepartum (0.54% DM of TMR) on mRNA gene expression profiles of placental samples of Holstein cows. Seventy multiparous Holstein cows were randomly assigned to 1 of 2 dietary treatments, consisting of TMR top-dressed with RPL (PRE-L) or without (control, CON), fed from 27 ± 5 d prepartum until calving. After natural delivery (6.87 ± 3.32 h), placentas were rinsed with physiological saline (0.9% sodium chloride solution) to clean any dirtiness from the environment and weighed. Then, 3 placentomes were collected, one from each placental region (cranial, central, and caudal), combined and flash-frozen in liquid nitrogen to evaluate the expression of transcripts and proteins related to protein metabolism and inflammation. Placental weights did not differ from cows in PRE-L (15.5 ± 4.03 kg) and cows in CON (14.5 ± 4.03 kg). Feeding RPL prepartum downregulated the expression of NOS3 (nitric oxide synthase 3), involved in vasodilation processes, and SOD1, which encodes the enzyme superoxide dismutase, involved in oxidative stress processes. Additionally, feeding RPL prepartum upregulated the expression of transcripts involved in energy metabolism (SLC2A3, glucose transporter 3; and PCK1, phosphoenolpyruvate carboxykinase 1), placental metabolism and cell proliferation (FGF2, fibroblast growth factor 2; FGF2R, fibroblast growth factor 2 receptor; and PGF, placental growth factor), Met metabolism (MAT2A, methionine adenosyltransferase 2-α), and tended to upregulate IGF2R (insulin-like growth factor 2 receptor). Placental FGF2 and LRP1 (low-density lipoprotein receptor-related protein 1) protein abundance were greater for cows that received RPL prepartum than cows in CON. In conclusion, feeding RPL to prepartum dairy cows altered uteroplacental expression of genes and proteins involved in cell proliferation, and in metabolism and transport of glucose. Such changes are illustrated by increased expression of SLC2A3 and PCK1 and increased protein abundance of FGF2 and LRP1 in uteroplacental tissue of cows consuming RPL.

Lychnophora ericoides Mart. (Brazilian arnica) ethanol extract accelerates the skin wound healing process: Evidence for its mechanism of action.

BACKGROUND: Lychnophora ericoides Mart, also known as the Brazilian arnica or fake arnica, belongs to the Asteraceae family. Leaves and roots are used in alcoholic and hydroalcoholic preparations for the treatment of wounds, inflammation, and pain. PURPOSE: The present study aimed to investigate the effects of L. ericoides ethanolic extract (EELE) on cutaneous wound healing and the mechanisms of action involved. METHODS: A total of 72 C57BL/6 mice were randomly divided into four groups of six animals each. An excisional wound was made in the dorsal region of each mouse. The test groups were topically treated with the vehicle, a positive control commercial reference drug, EELE ointment (5%), and EELE ointment (10%). The treatments were applied over 14 days. The wound area was measured every two days to verify the wound closure kinetics. On days 3, 7, and 14 the wound tissue samples were processed for Hematoxylin and Eosin, Masson-Trichrome, and Toluidine blue staining. The expression of renin-angiotensin system (RAS) components, the vascular growth factor-A (VEGF-A), the basic fibroblast growth factor (FGF-2), and type I collagen genes were evaluated. Phytochemical analyses were performed using HPLC-DAD and HPLC-MS/MS. RESULTS: The EELE (10%) significantly reduced the wound area compared to the treatments used for the other groups. Histological analysis demonstrated that wounds treated with L. ericoides for 14 days developed improved anatomical skin features, healed with hair follicles and sebaceous glands, increased collagen production and angiogenesis, and decreased the number of mast cells at the injury site. Real-time PCR data demonstrated that groups treated with EELE (10%) showed increased Type I collagen, VEGF-A, FGF-2, and AT1R and decreased ACE II and receptor MAS. The healing action of L. ericoides may be related to the presence of phenolic compounds, such as phenolic acids, chlorogenic acid derivatives, and C-glycoside flavonoids. CONCLUSION: Topical treatment with EELE increases important factors for wound healing: FGF, VEGF, collagen formation, and the expression of the proliferative axis of the renin-angiotensin system. For the first time, the present study shows the healing action of L. ericoides at the molecular level in an animal model. This process can be used as an alternative therapy for wound healing and the development of herbal therapy.

Effect of acupuncture on the opening time of implantation window and endometrial receptivity in controlled ovarian hyperstimulation rats during peri-implantation period.

Purpose: To investigate the effect of acupuncture for improving the pregnancy rate of COH rats from the viewpoint of regulating the opening time of the implantation window and endometrial receptivity. Methods: Experimental rats were randomly divided into normal group (N), model group (M) and acupuncture group(A), and samples were collected on Day 4, 5 and 6 after mating. COH rats were treated with acupuncture at SP6, LR3, and ST36 once a day for 7 times. The pinopodes were observed under a scanning electron microscope. Serum estrogen and progesterone levels were measured via ELISA. The protein and mRNA levels of estrogen receptor (ER), progesterone receptor (PR), leukemia inhibitory factor (LIF), integrin ß3, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2) in the endometrium were evaluated via West-blot, immunohistochemistry, and PCR. Results: Compared with group N, the pregnancy rate of group M was significantly decreased (P<0.05), and the abnormal serum hormone levels and implantation window advancement were observed. Compared with group M, the pregnancy rate of group A was significantly increased (P<0.05), the supraphysiological serum progesterone levels were restored to normalcy (P<0.05), and the advanced implantation window was restored to a certain extent. Further, the abnormal ER, PR, LIF, integrin ß3, VEGF, and FGF-2 expression levels of the endometrium got recovered to varying degrees. Conclusion: Acupuncture may restore the estrogen and progesterone balance in COH rats and the forward shift of the implantation window to a certain extent, improving the endometrial receptivity and finally improving the pregnancy rate of COH rats.

YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

BACKGROUND: Ocular neovascularization is a leading cause of blindness. Retinal microglia have been implicated in hypoxia-induced angiogenesis and vasculopathy, but the underlying mechanisms are not entirely clear. Lactylation is a novel lactate-derived posttranslational modification that plays key roles in multiple cellular processes. Since hypoxia in ischemic retinopathy is a precipitating factor for retinal neovascularization, lactylation is very likely to be involved in this process. The present study aimed to explore the role of lactylation in retinal neovascularization and identify new therapeutic targets for retinal neovascular diseases. RESULTS: Microglial depletion by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 suppresses retinal neovascularization in oxygen-induced retinopathy. Hypoxia increased lactylation in microglia and accelerates FGF2 expression, promoting retinal neovascularization. We identify 77 sites of 67 proteins with increased lactylation in the context of increased lactate under hypoxia. Our results show that the nonhistone protein Yin Yang-1 (YY1), a transcription factor, is lactylated at lysine 183 (K183), which is regulated by p300. Hyperlactylated YY1 directly enhances FGF2 transcription and promotes angiogenesis. YY1 mutation at K183 eliminates these effects. Overexpression of p300 increases YY1 lactylation and enhances angiogenesis in vitro and administration of the p300 inhibitor A485 greatly suppresses vascularization in vivo and in vitro. CONCLUSIONS: Our results suggest that YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression. Targeting the lactate/p300/YY1 lactylation/FGF2 axis may provide new therapeutic targets for proliferative retinopathies.

Quercetin relieves coronary atherosclerosis via regulating fibroblast growth factor 2.

This study aims to investigate the role of quercetin in coronary atherosclerosis and explore its possible mechanisms. Hematoxylin-eosin (H&E), immunohistochemical (IHC), and aniline blue staining were used to analyze the pathological changes in the cross-section of the aorta. Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), Swiss Target Prediction, and PubChem were utilized to predict and screen the bioactive ingredients of traditional Chinese medicine (Huanglian, Yuxingcao, and Jinyinhua) for coronary atherosclerosis. Inflammatory factors and vascular protection parameters were quantitatively detected using ELISA and western blot. The proliferation and migration of vascular smooth muscle cells (VSMC) were evaluated using the Cell Counting Kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), and wound healing assays. The targets of quercetin were predicted using DisGeNET, Matascape, SWISSMODEL, cellular thermal shift assay (CETSA), and fluorescence titrimetric methods. Based on our findings, quercetin was identified as the active component of Huanglian, Yuxingcao, and Jinyinhua that exerted a positive effect on coronary atherosclerosis. In vivo and in vitro data demonstrated that quercetin improved the pathological changes in model mice and inhibited the proliferation, migration, and inflammatory response of VSMC cells. Specifically, we found that fibroblast growth factor 2 (FGF2) is a direct target of quercetin, and overexpression of FGF2 attenuated the anti-atherosclerosis function of quercetin. Overall, our study confirms the functional role of the quercetin-FGF2 axis in the progression of coronary atherosclerosis, providing a potential target for its treatment.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Priming with a Combination of FGF2 and HGF Restores the Impaired Osteogenic Differentiation of Adipose-Derived Stem Cells.

Classical aging-associated diseases include osteoporosis, diabetes, hypertension, and arthritis. Osteoporosis causes the bone to become brittle, increasing fracture risk. Among the various treatments for fractures, stem cell transplantation is currently in the spotlight. Poor paracrine/differentiation capacity, owing to donor age or clinical history, limits efficacy. Lower levels of fibroblast growth factor 2 (FGF2) and hepatocyte growth factor (HGF) are involved in cell repopulation, angiogenesis, and bone formation in the elderly ADSCs (ADSC-E) than in the young ADSCs (ADSC-Y). Here, we study the effect of FGF2/HGF priming on the osteogenic potential of ADSC-E, determined by calcium deposition in vitro and ectopic bone formation in vivo. Age-induced FGF2/HGF deficiency was confirmed in ADSCs, and their supplementation enhanced the osteogenic differentiation ability of ADSC-E. Priming with FGF2/HGF caused an early shift of expression of osteogenic markers, including Runt-related transcription factor 2 (Runx-2), osterix, and alkaline phosphatase (ALP) during osteogenic differentiation. FGF2/HGF priming also created an environment favorable to osteogenesis by facilitating the secretion of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF). Bone tissue of ADSC-E origin was observed in mice transplanted with FGF/HGF-primed ADSC-E. Collectively, FGF2/HGF priming could enhance the bone-forming capacity in ADSC-E. Therefore, growth factor-mediated cellular priming can enhance ADSC differentiation in bone diseases and thus contributes to the increased efficacy in vivo.

FGF2 positively regulates osteoclastogenesis via activating the ERK-CREB pathway.

Fibroblast growth factor 2 (FGF2) plays crucial roles in the growth and development of several tissues. However, its function in bone homeostasis remains controversial. Here, we found that exogenous FGF2 supplementation inhibited the mineralization of bone marrow stromal cells (BMSCs), at least partially, via up-regulating the gene expression of osteoclastogenesis. The FGF receptor (FGFR) allosteric antagonist SSR128129E modestly, whereas the FGFR tyrosine kinase inhibitor AZD4547 significantly antagonized the effects of FGF2. Mechanistically, FGF2 stimulated ERK phosphorylation, and the ERK signaling inhibitor PD325901 strongly blocked FGF2 enhancement of osteoclastogenesis. Moreover, the phosphorylation of CREB was also activated in response to FGF2, thereby potentiating the interaction of p-CREB with the promoter region of Rankl gene. Notably, FGF2-deficient BMSCs exhibited higher mineralization capability and lower osteoclastogenic gene expression. Correspondingly, FGF2-knockout mice showed increased bone mass and attenuated expression of osteoclast-related markers, which were associated with moderate inhibition of the ERK signaling. In conclusion, FGF2 positively regulates osteoclastogenesis via stimulating the ERK-CREB pathway. These findings establish the importance of FGF2 in bone homeostasis, hinting the potential use of FGF2/ERK/CREB specific inhibitors to fight against bone-related disorders, such as osteoporosis.

Iron Oxide Nanoparticles in Mesenchymal Stem Cell Detection and Therapy.

Mesenchymal stem cells (MSCs) exhibit regenerative and reparative properties. However, most MSC-related studies remain to be translated for regular clinical usage, partly due to challenges in pre-transplantation cell labelling and post-transplantation cell tracking. Amidst this, there are growing concerns over the toxicity of commonly used gadolinium-based contrast agents that mediate in-vivo cell detection via MRI. This urges to search for equally effective but less toxic alternatives that would facilitate and enhance MSC detection post-administration and provide therapeutic benefits in-vivo. MSCs labelled with iron oxide nanoparticles (IONPs) have shown promising results in-vitro and in-vivo. Thus, it would be useful to revisit these studies before inventing new labelling approaches. Aiming to inform regenerative medicine and augment clinical applications of IONP-labelled MSCs, this review collates and critically evaluates the utility of IONPs in enhancing MSC detection and therapeutics. It explains the rationale, principle, and advantages of labelling MSCs with IONPs, and describes IONP-induced intracellular alterations and consequent cellular manifestations. By exemplifying clinical pathologies, it examines contextual in-vitro, animal, and clinical studies that used IONP-labelled bone marrow-, umbilical cord-, adipose tissue- and dental pulp-derived MSCs. It compiles and discusses studies involving MSC-labelling of IONPs in combinations with carbohydrates (Venofer, ferumoxytol, dextran, glucosamine), non-carbohydrate polymers [poly(L-lysine), poly(lactide-co-glycolide), poly(L-lactide), polydopamine], elements (ruthenium, selenium, gold, zinc), compounds/stains (silica, polyethylene glycol, fluorophore, rhodamine B, DAPI, Prussian blue), DNA, Fibroblast growth Factor-2 and the drug doxorubicin. Furthermore, IONP-labelling of MSC exosomes is reviewed. Also, limitations of IONP-labelling are addressed and methods of tackling those challenges are suggested.

Culture supplementation of bFGF, GDNF, and LIF alters in vitro proliferation, colony formation, and pluripotency of neonatal porcine germ cells.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7 days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10 ng/mL bFGF and 10 ng/mL bFGF + 100 ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on the growth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.

Basic fibroblast growth factor inhibits aortic valvular interstitial cells calcification via Notch1 pathway.

Calcific aortic valve disease (CAVD) is an active pathological process mediated by abnormal activation and transdifferentiation of valvular interstitial cells (VICs). The present study aims to investigate the function and underlying mechanism of the basic fibroblast growth factor (BFGF) on osteogenic differentiation of VICs. Porcine VICs cultured with osteogenic induction medium are supplemented with or without BFGF. Morphology of VICs is identified by fluorescein isothiocyanate-labeled phalloidin, the cell viability is assessed by the cell counting kit-8 method, and protein and mRNA expression level of osteogenic differentiation markers, including Runx2, osteopontin, and Sp7, are verified by western blot analysis and quantitative real-time PCR, respectively. RNA sequencing is used to identify changes in gene profiles. Alizarin Red S staining is used to measure calcium deposition. The results demonstrate that the content of calcium deposition and the expression level of osteogenic markers are downregulated by supplementing BFGF. Notch1 signaling pathway is extracted as a candidate target after bioinformatics analysis by RNA sequencing. The transfection of si-Notch1 abolishes the calcification inhibitory effect of BFGF. Taken together, our findings shed the light on the mechanism and potential therapeutics of BFGF for CAVD.

Myogenic Differentiation of iPS Cells Shows Different Efficiency in Simultaneous Comparison of Protocols.

Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-ß and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.

Endogenous FGF-2 levels impact FGF-2/BMP-2 growth factor delivery dosing in aged murine calvarial bone defects.

Bone repair in elderly mice has been shown to be improved or negatively impacted by supplementing the highly osteogenic bone morphogenetic protein-2 (BMP-2) with fibroblast growth factor-2 (FGF-2). To better predict the outcome of FGF-2 supplementation, we investigated whether endogenous levels of FGF-2 play a role in optimal dosing of FGF-2 for augmenting BMP-2 activity in elderly mice. In vivo calvarial bone defect studies in Fgf2 knockout mice with wildtype controls were conducted with the growth factors delivered in a highly localized manner from a biomimetic calcium phosphate/polyelectrolyte multilayer coating applied to a bone graft substitute. Endogenous FGF-2 levels were measured in old mice versus young and found to decrease with age. Optimal dosing for improving bone defect repair correlated with levels of endogenous FGF-2, with a larger dose of FGF-2 required to have a positive effect on bone healing in the Fgf2 knockout mice. The same dose in wildtype old mice, with higher levels of FGF-2, promoted chondrogenesis and increased osteoclast activity. The results suggest a personalized medicine approach, based on a knowledge of endogenous levels of FGF-2, should guide FGF-2 supplementation in order to avoid provoking excessive bone resorption and cartilage formation, both of which inhibited calvarial bone repair.

Selenium nanoparticles with various morphology for antiangiogenesis through bFGF-mediated P13K/AKT signaling pathways.

Selenium nanoparticles (Se NPs) have potential antitumor activity and immune properties. However, the mechanism between its antitumor activity and nanoparticle morphology has not been evaluated. Therefore, a simple method was used to synthesize three special shapes of Se NPs, which are fusiform, flower and spherical. Compared with fusiform selenium nanoparticles (Se NPs (S)) and flower-shaped selenium nanoparticles (Se NPs (F)), spherical selenium nanoparticles (Se NPs (B)) have better cell absorption effect and stronger antitumor activity. HRTEM showed that Se NPs (B) entered the nucleus through endocytosis and inhibited tumor angiogenesis by targeting basic fibroblast growth factor (bFGF). Se NPs (B) can competitively inhibit the binding of bFGF to fibroblast growth factor receptor through direct binding to bFGF, down-regulate the expression of bFGF in human umbilical vein endothelial cells (HUVEC), and significantly reduce the MAPK/Erk and P13K/AKT pathways activation of signaling molecules to regulate HUVEC cell migration and angiogenesis. These findings indicate that Se NPs have a special role in antitumor angiogenesis. This research provides useful information for the development of new strategies for effective drug delivery nanocarriers and therapeutic systems.

Mutual influence of selenium nanoparticles and FGF2-STAB® on biocompatible properties of collagen/chitosan 3D scaffolds: in vitro and ex ovo evaluation.

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection.

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor ß2 (TGF ß2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes' expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Saikosaponin d downregulates microRNA-155 and upregulates FGF2 to improve depression-like behaviors in rats induced by unpredictable chronic mild stress by negatively regulating NF-κB.

Saikosaponin d (SSd) is a traditional Chinese medicine that has been widely used in depression treatment. Given the lack of studies demonstrating the underlying mechanism of action of SSd in depression, the presented study was conducted with aims of investigating the effect of SSd on rats with depression-like behaviors induced by unpredicted chronic mild stress (UCMS) and its underlying molecular mechanism. To investigate the effect of SSd on depression, rat models with depression-like behaviors were established through 3-week exposure to UCMS, followed by administration of 10 mg/kg fluoxetine, 0.75 mg/kg SSd, 1.50 mg/kg SSd, or 10 mg/kg caffeic acid phenethyl ester (CAPE). The depression-like behaviors of rats were evaluated by sucrose preference test, open field test, forced swimming test, and tail suspension test. Afterwards, the regulatory relationship among nuclear factor-κB (NF-κB), microRNA (miR)-155 and fibroblast growth factor 2 (FGF2) were detected by dual-luciferase reporter gene assay and ChIP. RT-qPCR and Western blot analysis was conducted to determine the expression of genes and proteins. Finally, hippocampal neurons were extracted from modeled rats and transfected with miR-155 mimic, miR-155 inhibitor, NF-κB overexpression plasmid, or siRNA against NF-κB. The results showed that the depression-like behaviors induced by UCMS in rats was successfully attenuated by SSd. In hippocampal neurons of rats treated with SSd, NF-κB was significantly downregulated while FGF2 was significantly upregulated. NF-κB targets miR-155 and negatively regulates the expression of FGF2. NF-κB knockdown resulted in reduced depression-like behaviors of rats. These findings provide evidence that SSd could ameliorate depression-like behaviors in the rats treated with UCMS by downregulating NF-κB and miR-155, and upregulating FGF2.

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2.

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.