Development and Optimization of Freeze-Dried Eye Drops Derived from Plasma Rich in Growth Factors Technology

Purpose: To investigate whether plasma rich in growth factors (PRGF) eye drops maintain their biological potential after a freeze drying process. The addition of a lyopro-tectant like trehalose was also evaluated. Methods: Blood from three healthy donors was collected to obtain eye drops by PRGF technology. The resultant eye drops were divided in four groups: PRGF, freeze-dried PRGF (PRGF lyo), and PRGF lyophilized mixed with 2,5% trehalose (PRGF lyo+2.5T) or 5% trehalose (PRGF lyof+5T). Chemical and biological characteristics were evaluated. Photorefractive keratectomy was performed on C57BL/6 mice which were divided in three treatment groups: control, PRGF, and PRGF lyo. Corneal wound healing and haze formationwere evaluated macroscopically. Eyeswere collected at 1, 2, 3, and 7 days after surgery, and were processed for histologic studies. Results: The pH values of PRGF samples increased significantly after the lyophilization process. Osmolarity levels increased significantly in PRGF samplesmixed with trehalose in comparison with PRGF samples without protectants. The freeze drying process maintained growth factors levels as well as the biological properties of PRGF eye drops even without the use of lyoprotectants. PRGF lyo treatment significantly decreased the re-epithelialization time and haze formation in photorefractive keratectomy-treated corneas regarding PRGF and control groups. Furthermore, the PRGF lyo group significantly decreased the number of smooth muscle actin-positive cells in comparison with the control group at each time of the study and at days 2 and 3 in the PRGF group. Conclusions: The freeze drying process preserves the protein and growth factor content as well as the biological properties of PRGF eye drops, even without the use of protectants. Freeze-dried PRGF eye drops accelerate corneal tissue regeneration after photorefractive keratectomy in comparison with the control group. Translational Relevance: Our study shows the feasibility to preserve the biological capability of PRGF eye drops as freeze-dried formulation, avoiding the addition of protectants.This study received funding from the Basque Country Government, within the Elkartek program, phase I. Support program for collaborative research in strategic area, within the project named SINET (reference KK-2018/00048

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis

Background: The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear. Methods: The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 mu m in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence. Results: Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells. Conclusion: 3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.Supported in part by Pharmakine S. L., and by grants from the CICYT of the Spanish government (SAF2006-09341), and the Basque Country Government (IT-487-07

Differential profile of protein expression on human keratocytes treated with autologous serum and plasma rich in growth factors (PRGF)

Purpose The main objective of this study is to compare the protein expression of human keratocytes treated with Plasma rich in growth factors (PRGF) or autologous serum (AS) and previously induced to myofibroblast by TGF-beta 1 treatment. Methods Blood from healthy donor was collected and processed to obtain AS and PRGF eye drops. Blood derivates were aliquoted and stored at-80 degrees C until use. Keratocyte cells were pretreated for 60 minutes with 2.5 ng/ml TGF-beta 1. After that, cells were treated with PRGF, AS or with TGF-beta 1 (control). To characterize the proteins deregulated after PRGF and AS treatment, a proteomic approach that combines 1D-SDS-PAGE approach followed by LC-MS/MS was carried out. Results Results show a catalogue of key proteins in close contact with a myofibroblastic differentiated phenotype in AS treated-cells, whereas PRGF-treated cells show attenuation on this phenotype. The number of proteins downregulated after PRGF treatment or upregulated in AS-treated cells suggest a close relationship between AS-treated cells and cytoskeletal functions. On the other hand, proteins upregulated after PRGF-treatment or downregulated in AS-treated cells reveal a greater association with processes such as protein synthesis, proliferation and cellular motility. Conclusion This proteomic analysis helps to understand the molecular events underlying AS and PRGF-driven tissue regeneration processes, providing new evidence that comes along with the modulation of TGF-beta 1 activity and the reversion of the myofibroblastic phenotype by PRGF.This study was fully supported by BTI Biotechnology Institute, a dental implant company that investigates in the fields of oral implantology and PRGF-Endoret technology. MF and FM received a salary as scientists from BTI Biotechnology Institute. EA is the Scientific Director and president of BTI Biotechnology Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Plasma Rich in Growth Factors Membrane as Coadjuvant Treatment in the Surgery of Ocular Surface Disorders

To evaluate the safety and efficacy of the surgical use of plasma rich in growth factors fibrin membrane (mPRGF) in different ocular surface pathologies.Fifteen patients with different corneal and conjunctival diseases were included in the study. Patients were grouped according to the use of mPRGF as graft (corneal and/or conjunctival) or dressing; they were also grouped according to the surgical subgroup of intervention (persistent corneal ulcer [PCU], keratoplasty, superficial keratectomy, corneal perforation, and pterygium). Best corrected visual acuity, intraocular pressure (IOP), inflammation control time (ICT), mPRGF AT (PRGF membrane absorption time), and the healing time of the epithelial defect (HTED) were evaluated throughout the clinical follow-up time. Safety assessment was also performed reporting all adverse events.mPRGF showed a total closure of the defect in 13 of 15 patients (86.7%) and a partial closure in 2 patients (13.3%). The mean follow-up time was 11.14.2 (4.8-22.8) months, the mean ICT was 2.5 +/- 1.1 (1.0-4.0) months, the mean mPRGF AT was 12.4 +/- 2.0 (10.0-16.0) days, and for the global HTED the mean was 2.9 +/- 1.2 (1-4.8) months. Results showed an improvement in BCVA in all patients, with an overall improvement of 2.9 in Vision Lines. The BCVA significantly improved (P.05) throughout the clinical follow-up time. No adverse events were reported after mPRGF use.The mPRGF is effective and safe as coadjuvant treatment in surgeries related with ocular surface disorders, being an alternative to the use of amniotic membrane. The mPRGF accelerates tissue regeneration after ocular surface surgery thus minimizing inflammation and fibrosis.This study received funding from the Ministry of Economy and Competitiveness of the Spanish Government, within the project denominated SURFEYE (reference RTC-2014-2375-1)

The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation

The ability to ensure continuous availability of energy despite highly variable supplies in the environment is a major determinant of the survival of all species. In higher organisms, including mammals, the capacity to efficiently store excess energy as triglycerides in adipocytes, from which stored energy could be rapidly released for use at other sites, was developed. To orchestrate the processes of energy storage and release, highly integrated systems operating on several physiological levels have evolved. The adipocyte is no longer considered a passive bystander, because fat cells actively secrete many members of the cytokine family, such as leptin, tumor necrosis factor-alpha, and interleukin-6, among other cytokine signals, which influence peripheral fuel storage, mobilization, and combustion, as well as energy homeostasis. The existence of a network of adipose tissue signaling pathways, arranged in a hierarchical fashion, constitutes a metabolic repertoire that enables the organism to adapt to a wide range of different metabolic challenges, such as starvation, stress, infection, and short periods of gross energy excess