Combination of leukocyte and platelet-rich fibrin and demineralized bovine bone graft enhanced bone formation and healing after maxillary sinus augmentation: a randomized clinical trial.

BACKGROUND AND OBJECTIVE: The resorption of alveolar ridge bone and maxillary sinus pneumatization are challenges to implant-supported prosthetic rehabilitation. Bone regeneration using bone substitutes and growth factors are alternatives for maxillary sinus augmentation (MSA). Therefore, we sought to evaluate the effects of the association between leukocyte and platelet-rich fibrin (L-PRF) and deproteinized bovine bone mineral (DBBM) in MSA procedures. MATERIALS AND METHODS: Thirty-six maxillary sinuses from 24 individuals were included in this randomized clinical trial. The maxillary sinuses were randomly grafted with LPRF and DBBM (test group) or grafted only with DBBM (positive control). Dental implants were installed in the test group following two periods of evaluation: after 4 (DBBM+LPRF4) and 8 (DBBM+LPFR8) months of sinus graft healing, while the control group received implants only after 8 months. Cone beam computed tomography (CBCT) was taken 1 week after surgery (T1) and before implant placement (T2). Bone samples were collected during implant placement for histomorphometric and immunohistochemical (IHC) analysis. The primary implant stability was assessed by resonance frequency analysis. RESULTS: CBCT analysis demonstrated a significant decrease in bone volume from T1 to T2 in all groups without differences among them. Histologically, the test group showed significantly increase in bone neoformation in both periods of evaluation (LPRF+DBBM4: 44.70±14.01%; LPRF+DBBM8: 46.56±12.25%) compared to the control group (32.34±9.49%). The control group showed the highest percentage of residual graft. IHC analysis showed increased staining intensity of osteocalcin (OCN), vascular endothelial growth factor (VEGF), and runt related transcription factor 2 (RUNX-2) in LPRF+DBBM4 group, and osteopontin (OPN) in the L-PRF+DBBM8. Primary implant stability was successfully achieved (above 60 in implant stability quotient) in all the evaluated groups. CONCLUSION: Combination of L-PRF and DBBM increased and accelerated new bone formation allowing early implant placement probably due to the higher protein expression of RUNX2, VEGF, OCN, and OPN. These data suggest that the use of L-PRF might be an interesting alternative to use in combination with DBBM for augment the maxillary sinuses allowing the installation of appropriate length implants in shorter period of time. CLINICAL RELEVANCE: This study showed improvement in bone neoformation and accelerated healing when associating L-PRF and DBBM for maxillary sinus augmentation procedures. TRIAL REGISTRATION: This study was registered before participant recruitment in Brazilian Registry of Clinical Trials (ReBEC - RBR-95m73t).

Lactobacillus plantarum CQPC02 intervenes in mouse lupus nephritis by regulating the NF-κB signaling pathway.

A strain of Lactobacillus plantarum CQPC02 (LP-CQPC02) isolated from naturally fermented kimchi was utilized in this investigation. In order to construct an animal model of lupus nephritis, pristane was used. We then used a kit to identify markers in mouse blood and tissues and a quantitative polymerase chain reaction (qPCR) to measure the expression of genes associated to nuclear factor kappa-B (NF-κB) in mouse kidney tissue. According to the results of the experiments, oral administration of LP-CQPC02 LP-CQPC02 may lessen the lupus nephritis-related rise in urine protein as well as the cytokine levels that were rising in the serum and renal tissues, including IL-6, IL-12, tumor necrosis factor alpha, and interferon. Additionally, in mice with nephritis, LP-CQPC02 can lower serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol (TC), triglyceride (TG), and raise total protein (TP) and albumin (ALB) levels. In mice with nephritis, LP-CQPC02 can also reduce the positive rate of double-stranded deoxyribonucleic acid (dsDNA). Pathological sections were examined, and it was shown that LP-CQPC02 can lessen tissue damage such incomplete glomerular morphology and inflammatory infiltration brought on by nephritis. In the kidneys of mice with lupus nephritis, LP-CQPC02 can upregulate the expression of inhibitor of NF-κB (IκB-α), downregulate the expression of NF-κB, transforming growth factor-ß1 (TGF-ß1), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Lactobacillus plantarum CQPC02 has been confirmed to have an intervention effect on nephritis in mice and has the potential as a probiotic.

Atorvastatin shows antitumor effect in hepatocellular carcinoma development by inhibiting angiogenesis via TGF-ß1/pERK signaling pathway.

Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins may reduce HCC incidence. Its antitumor activities may be mediated by disrupting several hepatocarcinogenic pathways. To evaluate in vivo and in vitro the antiproliferative and antiangiogenic action of atorvastatin (AT) in the development of HCC as well as its mechanisms of action. In vivo model: hexachlorobenzene (HCB) was used to promote the development of HCC in Balb/C nude mice. Number of hepatic tumor, liver cell proliferation parameters (proliferating cell nuclear antigen, PCNA), angiogenesis, and VEGF levels were analyzed. In vitro model: Hep-G2 and Ea-hy926 cells were used to evaluate the effect of different doses of AT on HCB induced cell proliferation, migration, and vasculogenesis and to analyze proliferative parameters. In vivo: AT prevented liver growth and tumor development and inhibited PCNA, TGF-ß1, and pERK levels increase. AT prevented skin blood vessel formation. In vitro, AT prevented cell proliferation and migration as well as tubular formation in the endothelial cell line by inhibiting the MAPK ERK pathway. We were able to demonstrate the potential AT antiproliferative and antiangiogenic effects in an HCC model and the involvement of TGF-ß1 and pERK pathways.

Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion.

OBJECTIVE: This study aimed to evaluate the effects of systemic teriparatide on sutural bone formation after premaxillary suture expansion in rats. MATERIAL AND METHODS: Twenty Wistar male rats (8-10 weeks old) were randomly divided into two groups, namely, control (C, n=10) and teriparatide (T, n=10). An expansion force was applied to the maxillary incisors using helical spring for a seven-day expansion period, for both groups. On the eighth day, the rats were kept for a seven-day consolidation period, and then 60 µg/kg teriparatide (once a day) was administered to group T subcutaneously for seven days. Then, all the rats were sacrificed, and histological sections were stained with hemotoxylin-eosin for examination. Anti-osteonectin, anti-osteocalcin, anti-Vascular endothelial growth factor (VEGF) and anti-transforming growth factor beta (TGF-ß) were evaluated by immunohistochemical analysis in the midpalatal suture area. RESULTS: Histologically, the newly formed bone tissue was observed to be larger in group T than in group C. The number of immunoreactive osteoblasts for osteonectin, osteocalcin and VEGF antibodies was significantly higher in group T than in group C (p = 0.0001). The TGF-ß antibody showed a mild reaction in group T, but did not reach significance in comparison with group C (p ˃ 0.05). CONCLUSION: Systemic teriparatide application following the premaxillary expansion of the suture area may stimulate bone formation and add to the consolidation of the expansion in rats by regulating osteonectin, osteocalcin and VEGF.

Secretome from Human Mesenchymal Stem Cells-Derived Endothelial Cells Promotes Wound Healing in a Type-2 Diabetes Mouse Model.

Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton's jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.

Superior mesenteric ganglion neural modulation of ovarian angiogenesis, apoptosis and proliferation by the neuroactive steroid allopregnanolone.

Allopregnanolone (ALLO), a potent neuroactive steroid, is synthesized and active in the peripheral nervous system. Previous studies have shown that ALLO participates in the central regulation of reproduction with effects on ovarian physiology, although there is little evidence for its ability to modulate peripheral tissues. The present study aimed to determine whether ALLO, administered to an ex vivo system that comprises the superior mesenteric ganglion (SMG), the ovarian nervous plexus (ONP) and the ovary (O), or to the denervated ovary (DO), was able to modify ovarian apoptosis, proliferation and angiogenesis. For this purpose, the SMG-ONP-O system and DO were incubated during 120 min at 37°C, in the presence of two ALLO doses (0.06 µm and 6 µm). The intrinsic and extrinsic pathways of apoptosis were analyzed. Incubation of the SMG-ONP-O system with ALLO 0.06 µm led to an increase in the BAX/BCL-2 ratio and a reduction of FAS-L mRNA levels. ALLO 6 µm induced a decrease of FAS-L levels. Incubation of DO with ALLO 0.06 µm reduced FAS-L, whereas ALLO 6 µm significantly increased it. Cyclin D1 mRNA was measured to evaluate proliferation. Treatment with ALLO 6 µm increased proliferation in both SMG-ONP-O and DO. ALLO 0.06 µm produced an increase of Cyclin D1 in DO only. Administration of either ALLO dose led to a higher ovarian expression of vascular endothelial growth factor in the SMG-ONP-O system, but a lower one in the DO system. ALLO 6 µm induced ovarian sensitization to GABA by increasing GABAA receptor expression. In conclusion, ALLO participates in the peripheral neural modulation of ovarian physiology. It can also interact directly with the ovarian tissue, modulating key mechanisms involved in normal and pathological processes in a dose-dependent manner.

Hypoxia Supports Differentiation of Terminally Exhausted CD8 T Cells.

Hypoxia, angiogenesis, and immunosuppression have been proposed to be interrelated events that fuel tumor progression and impair the clinical effectiveness of anti-tumor therapies. Here we present new mechanistic data highlighting the role of hypoxia in fine-tuning CD8 T cell exhaustion in vitro, in an attempt to reconcile seemingly opposite evidence regarding the impact of hypoxia on functional features of exhausted CD8 T cells. Focusing on the recently characterized terminally-differentiated and progenitor exhausted CD8 T cells, we found that both hypoxia and its regulated mediator, vascular endothelial growth factor (VEGF)-A, promote the differentiation of PD-1+ TIM-3+ CXCR5+ terminally exhausted-like CD8 T cells at the expense of PD-1+ TIM-3- progenitor-like subsets without affecting tumor necrosis factor (TNF)-α and interferon (IFN)-γ production or granzyme B (GZMB) expression by these subpopulations. Interestingly, hypoxia accentuated the proangiogenic secretory profile in exhausted CD8 T cells. VEGF-A was the main factor differentially secreted by exhausted CD8 T cells under hypoxic conditions. In this sense, we found that VEGF-A contributes to generation of terminally exhausted CD8 T cells during in vitro differentiation. Altogether, our findings highlight the reciprocal regulation between hypoxia, angiogenesis, and immunosuppression, providing a rational basis to optimize synergistic combinations of antiangiogenic and immunotherapeutic strategies, with the overarching goal of improving the efficacy of these treatments.

Heterotopic autotransplantation of ovarian tissue in a large animal model: Effects of cooling and VEGF.

Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.

The NO-donor MPC-1011 stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis. METHODS: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging. RESULTS: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin-/CD31+/CXCR4+ cells were increased by 92.0% and Lin-/VEGFR2+/CXCR4+ cells by 76.8% as compared to placebo. CONCLUSIONS: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.

Expression and Characterization of Human Vascular Endothelial Growth Factor Produced in SiHa Cells Transduced with Adenoviral Vector.

The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35 kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.

Induction of typical and atypical neurogenesis in the adult substantia nigra after mouse embryonic stem cells transplantation.

Neurogenesis in the substantia nigra (SN) has been a controversial issue. Here we report that neurogenesis can be induced in the adult rodent SN by transplantation of embryoid body cells (EBCs) derived from mouse embryonic stem cells. The detection of Sox2+ dividing (BrdU+) putative host neural precursor cells (NPCs) between 1 and 6 days post-transplantation (dpt) supported the neurogenic capacity of the adult SN. In agreement with the awakening of NPCs by EBCs, only host cells from implant-bearing SN were able to generate neurosphere-like aggregates in the presence of Egf and Fgf2. Later, at 15 dpt, a significant number of SN Dcx+ neuroblasts were detected. However, a continuous BrdU administration after transplantation showed that only a fraction (about 20-30%) of those host Dcx+ progeny derived from dividing cells and few BrdU+ cells, some of them NeuN+, survived up to 30 dpt. Unexpectedly, 25-30% of Dcx+ or Psa-Ncam+ cells at 15 dpt displayed astrocytic markers such as Gfap and S100b. Using a genetic lineage tracing strategy, we demonstrated that a large proportion of host Dcx+ and/or Tubb3+ neuroblasts originated from Gfap+ cells. Remarkably, new blood vessels formed in association with the neurogenic process that, when precluded, caused a reduction in neuroblast production. Accordingly, two proteins secreted by EBCs, Fgf2 and Vegf, were able to promote the emergence of Dcx+/Psa-Ncam+, Tubb3+ and NeuN+/BrdU+ cells in vivo in the absence of EBCs. We propose that the adult SN is a mostly silent neurogenic niche with the ability to generate new neurons by typical and atypical mechanisms.

Immunohistological study of the effect of vascular Endothelial Growth Factor on the angiogenesis of mature root canals in rat molars.

Tissue bioengineering has been applied to Endodontics to seek a more biological treatment. The presence of blood vessels is crucial for cell nutrition during tissue formation. Objective This study analysed the application of vascular endothelial growth factor (VEGF) in the angiogenesis of mature root canals. Material and methods Upper first molars of twelve 13-week old Wistar male rats were used. The root pulp of the mesiobuccal canal was removed and the root canal instrumented with K-files up to size #25. Periapical bleeding was induced into the root canal by introducing a #15 K-file beyond the apex. The teeth on the right side of the arch were filled up with blood clot (G1), whereas those on the left side were filled up with blood clot plus 50 ng/ml of VEGF (G2). Teeth were sealed with light-curing glass-ionomer cement and the animals were sacrificed after 60 days. The maxilla was dissected and fixed before obtaining serial sections for histological processing with haematoxylin-eosin (HE) and immunohistochemical factor-VIII. Immunohistochemical labelling was evaluated using scores for statistical analysis. Results Immunohistological analysis demonstrated the presence of angiogenesis in both groups, but with higher angiogenic maturation in G2 during the experimental period (p<0.05). HE staining showed connective tissue with absence of odontoblasts in all specimens. Conclusions It can be concluded that it is possible to obtain angiogenesis in mature root canals with or without the use of VEGF, although the latter tends to accelerate blood vessel formation.

In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex.

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.

Immunohistological study of the effect of vascular Endothelial Growth Factor on the angiogenesis of mature root canals in rat molars

Abstract Tissue bioengineering has been applied to Endodontics to seek a more biological treatment. The presence of blood vessels is crucial for cell nutrition during tissue formation. Objective This study analysed the application of vascular endothelial growth factor (VEGF) in the angiogenesis of mature root canals. Material and methods Upper first molars of twelve 13-week old Wistar male rats were used. The root pulp of the mesiobuccal canal was removed and the root canal instrumented with K-files up to size #25. Periapical bleeding was induced into the root canal by introducing a #15 K-file beyond the apex. The teeth on the right side of the arch were filled up with blood clot (G1), whereas those on the left side were filled up with blood clot plus 50 ng/ml of VEGF (G2). Teeth were sealed with light-curing glass-ionomer cement and the animals were sacrificed after 60 days. The maxilla was dissected and fixed before obtaining serial sections for histological processing with haematoxylin-eosin (HE) and immunohistochemical factor-VIII. Immunohistochemical labelling was evaluated using scores for statistical analysis. Results Immunohistological analysis demonstrated the presence of angiogenesis in both groups, but with higher angiogenic maturation in G2 during the experimental period (p<0.05). HE staining showed connective tissue with absence of odontoblasts in all specimens. Conclusions It can be concluded that it is possible to obtain angiogenesis in mature root canals with or without the use of VEGF, although the latter tends to accelerate blood vessel formation.

Caprine ovarian follicle requirements differ between preantral and early antral stages after IVC in medium supplemented with GH and VEGF alone or in combination.

The aim of the present study was to evaluate the effect of growth hormone (GH) and vascular endothelial growth factor (VEGF) added alone, sequentially or in combination, in the presence of insulin at physiological concentration (10 ng/mL) on the IVC of two different follicular categories: preantral (experiment 1; Exp.1) and early antral (experiment 2; Exp.2). Isolated follicles were individually cultured for 24 (Exp.1) and 18 days (Exp.2) in the following treatments: αMEM+ (Control), or Control medium supplemented with 50 ng/mL GH (GH), 100 ng/mL VEGF (VEGF), the combination of both (GH + VEGF), GH during the first 12 days and VEGF from Day 12 until the end of the culture (GH/VEGF) and vice versa (VEGF/GH). At the end of the culture, cumulus-oocyte complexes from in vitro-grown follicles were recovered and subjected to IVM. The following end points were evaluated: Follicle morphology, growth rates and antrum formation, production of estradiol, progesterone and testosterone, oocyte viability and meiotic stage, as well as relative expression of LHR, Amh, HAS2, PTGS2, CYP17, CYP19A1, and 3ßHSD. A considerable amount of viable fully grown oocytes were recovered after the IVC of early antral follicles in all treatments. Nevertheless, the GH treatment presented the highest percentage of fully grown oocytes (60%), mean oocyte diameter (117.74 ± 2.61 µm), and meiotic resumption (50%). Furthermore, GH treatment produced higher (P < 0.05) rates of metaphase II oocytes than all the other treatments, and similar LHR, Amh, and PTGS2 transcript levels to in vivo. Contrary to early antral follicles, preantral follicles were not affected by medium supplementation. In conclusion, the addition of GH to a culture medium containing physiological concentrations of insulin improves oocyte growth and maturation after the IVC of goat early antral follicles.

Effects of low-level laser therapy and epidermal growth factor on the activities of gingival fibroblasts obtained from young or elderly individuals.

This study evaluated the effects of low-level laser therapy (LLLT) and epidermal growth factor (EGF) on fibroblasts obtained from young and elderly individuals. Gingival fibroblasts from young (Y) and elderly (E) individuals were seeded in wells of 24-well plates with Dulbecco's modified Eagle's medium (DMEM) containing 10 % of fetal bovine serum (FBS). After 24 h, the cells were irradiated (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J/cm2) or exposed to EGF (100 µM). After 72 h, cells were evaluated for viability, migration, collagen and vascular endothelial growth factor (VEGF) synthesis, and gene expression of growth factors. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 5 %). Y and E fibroblasts irradiated with laser or exposed to EGF showed increased viability and collagen synthesis. Enhanced cell migration was observed for Y fibroblasts after both treatments, whereas only the LLLT stimulated migration of E cells. VEGF synthesis was higher for Y and E cells exposed to EGF, while this synthesis was reduced when E fibroblasts were irradiated. Increased gene expression of VEGF was observed only for Y and E fibroblasts treated with LLLT. Regardless of a patient's age, the LLLT and EGF applications can biostimulate gingival fibroblast functions involved in tissue repair.

Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors.

Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.

Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration.

The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy.

Influence of vascular endothelial growth factor and Cysteamine on in vitro bovine oocyte maturation and subsequent embryo development.

The objective of this study was to investigate the effect of VEGF and Cysteamine during in vitro maturation (IVM) of bovine oocytes on GSH content and developmental competence. For this purpose, experiments were designed to evaluate the effect of 0, 100, 300, and 500 ng/mL VEGF in IVM medium on: GSH content in oocytes and cumulus cells (Exp. 1) and subsequent embryo development (Exp. 2). Also, influence of adding 500 ng/mL VEGF and 100 µM Cysteamine to IVM medium on GSH content in oocytes and cumulus cells (Exp. 3) and oocyte developmental capacity (Exp. 4) were evaluated. Oocytes were matured in: a) Control; b) VEGF 0-3 h; c) Cysteamine 4-24 h; d) VEGF 0-3 h + Cysteamine 4-24 h; and e) VEGF + Cysteamine 24 h. The results showed that: i) VEGF did not alter GSH content in oocytes and cumulus cells; (ii) supplementation of 300 and 500 ng/mL VEGF increased blastocyst yield; (iii) the presence of VEGF + Cysteamine simultaneously during 24 h improved GSH content but not embryo development; and (iv) the presence of VEGF during the first 3 h + Cysteamine from 4 to 24 h increased GSH concentrations and subsequent embryo development. In conclusion, the addition of VEGF and Cysteamine in two sequential steps to maturation medium result in an improvement of cytoplasmic maturation, with a positive impact on oocyte developmental capacity by increasing the efficiency of in vitro blastocyst production. However, the effect was detrimental when both VEGF and Cysteamine were present during 24 of IVM.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.