Evaluating Membranes for Bone Regeneration

This work was supported by: 1)the Ministry of Economy and Competitiveness and European Regional Development Fund [Project MAT2017-85999-P MINECO/AEI/FEDER/UE],2) University of Granada/Regional Government of Andalusia Research Fund from Spain and European Regional Development Fund (A-BIO-157-UGR-18/FEDER).This research is part of M.T-O.’s PhD research study.Objectives:Maxillofacial bone defectsare the main hindering conditions for traditional dental implant strategies. Guided Bone Regeneration (GBR) is used to handle this situation. The principle of GBR is to use a membrane to prevent the colonization of soft tissue cells of the bone defect and favors the migration of osteogenic linages. Currentmembranes do not completely fulfill the requirements that an optimal membrane should have, sometimes resulting in non-predictable results. Thus, the need to develop an ideal membrane to perform this duty is clear. Recent developments in bio-manufacturing are driving innovations in membranes technology permitting the active participation of the membrane in the healing and regenerative process trough native tissue mimicking, drug-delivery and cells interaction, away from being a passive barrier. New membranes features need specific evaluation techniques, beyond the International Standard for membrane materials (last reviewed in 2004), being this the rationale for the present review.Nanotechnology application has completely shifted the way of analyzing structural characterization. New progresses on osteoimmmunomodulation have also switched the understanding of cells-membranes interaction. Data and Sources:To propose an updated protocol for GBR membranes evaluation, critical reading of therelevant published literature was carried out after a MEDLINE/PubMed database search. Conclusions:The main findings are that a potential active membrane should be assessed in its nanostructure, physicochemical and nanomechanical properties,bioactivityand antibacterial,osteoblasts proliferation, differentiation and mineralization. Immunomodulationtesting for macrophages recruitmentand M2 phenotype promotion in osteoblasts co-culture has to beachieved to completely analyze membranes/tissue interactions.Ministry of Economy and Competitiveness and European Regional Development Fund [Project MAT2017-85999-P MINECO/AEI/FEDER/UE]University of Granada/Regional Government of Andalusia Research Fund from Spain and European Regional Development Fund (A-BIO-157-UGR-18/FEDER

Mechanism and Consequences of The Impaired Hif-1α Response to Hypoxia in Human Proximal Tubular HK-2 Cells Exposed to High Glucose

Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at diferent levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These efects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetesinduced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative efects of the diabetic milieu on PTCThis work was supported by grants SAF2014-53218-R from the Spanish Ministerio de Ciencia e Innovación and RTI2018-095872-B-C21/ERDF by AEI. Coral Garcia-Pastor is the recipient of a FPU fellowship from the University of Alcala

Impact of bisphosphonates on the proliferation and gene expression of human fibroblasts

The aim of this study was to elucidate the role of fibroblasts in bisphosphonate-related osteonecrosis of the jaw (BRONJ), evaluating the effect of zoledronate, alendronate, and ibandronate on the proliferation of fibroblasts and on their expression of genes essential for fibroblast physiology. Human CCD-1064Sk epithelial fibroblast cells were incubated in culture medium with 10-5, 10-7, or 10-9 M zoledronate, alendronate, or ibandronate. The proliferative capacity of fibroblasts was determined by spectrophotometry (MTT) at 24 of culture. Real-time polymerase chain reaction (RT-PCR) was used to study the effects of BPs at a dose of 10-9 M on the expression of FGF, CTGF, TGF-β1, TGFβR1, TGFβR2, TGFβR3, DDR2, α-actin, fibronectin, decorin, and elastin. Fibroblasts proliferation was significantly increased at the lowest dose (10-9M) of each BP but was not affected at the higher doses (10-5 and 10-7M). The proliferation increase may be related to the rise in TGF-β1 and TGFβR1 expression detected after the treatment of cells with 10-9M of zoledronate, alendronate, or ibandronate. However, the expression of CTGF, DDR2, α-actin, fibronectin, and decorin decreased versus controls. The results of this in vitro study indicate that a very low BP dose (10-9 M) can significantly affect the physiology of fibroblasts, increasing their proliferative capacity and modulating the expression of multiple genes involved in their growth and differentiation

Effects of Therapeutic Doses of Celecoxib on Several Physiological Parameters of Cultured Human Osteoblasts

Non-steroidal anti-inflammatory drugs (NSAIDs), including cyclooxygenase-2 (COX-2)-selective NSAIDs, are associated with adverse effects on bone tissue. These drugs are frequently the treatment of choice but are the least studied with respect to their repercussion on bone. The objective of this study was to determine the effects of celecoxib on cultured human osteoblasts. Human osteoblasts obtained by primary culture from bone samples were treated with celecoxib at doses of 0.75, 2, or 5μM for 24 h. The MTT technique was used to determine the effect on proliferation; flow cytometry to establish the effect on cell cycle, cell viability, and antigenic profile; and real-time polymerase chain reaction to measure the effect on gene expressions of the differentiation markers RUNX2, alkaline phosphatase (ALP), osteocalcin (OSC), and osterix (OSX). Therapeutic doses of celecoxib had no effect on osteoblast cell growth or antigen expression but had a negative impact on the gene expression of RUNX2 and OSC, although there was no significant change in the expression of ALP and OSX. Celecoxib at therapeutic doses has no apparent adverse effects on cultured human osteoblasts and only inhibits the expression of some differentiation markers. These characteristics may place this drug in a preferential position among NSAIDs used for analgesic and anti-inflammatory therapy during bone tissue repair.This study was supported by research group BIO277 (Junta de Andalucía) and Department of Nursing (University of Granada). The work outlined in this article has been supported by the Spanish Ministry of Education under FPU fellowship reference FPU15-05635 and FPU16-04141

Mechanism and Consequences of The Impaired Hif-1alfa Response to Hypoxia in Human Proximal Tubular HK-2 Cells Exposed to High Glucose

Renal hypoxia and loss of proximal tubular cells (PTC ) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1? (HIF-1?) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1? regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1? degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1? with Hsp90, a known cause of proteasomal degradation of HIF-1?. Impaired HIF-1? regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1? regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1? degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.Ministerio de Ciencia e Innovació

Dexamethasone and doxycycline functionalized nanoparticles enhance osteogenic properties of titanium surfaces

Objectives: To evaluate the effect of doxycycline and dexamethasone doped nanoparticles covering titanium surfaces, on osteoblasts proliferation and differentiation. Methods: Doxycycline and dexamethasone doped polymeric nanoparticles were applied on titanium discs (Ti- DoxNPs and Ti-DexNPs). Undoped NPs and uncovered Ti discs were used as control. Human MG-63 osteoblast- like cells were cultured. Osteoblasts proliferation was tested by MTT assay. Alkaline phosphatase activity was analyzed. Differentiation gene expression was assessed by real-time quantitative polymerase chain reaction. Scanning Electron Microscopy was performed to assess osteoblasts morphology. Mean comparisons were con- ducted by ANOVA and Wilcoxon or Tukey tests (p < 0.05). Results: No differences in osteoblasts proliferation were found. Osteoblasts grown on Ti-DoxNPs significantly increased alkaline phosphatase activity. Doxycycline and dexamethasone nanoparticles produced an over-ex- pression of the main osteogenic proliferative genes (TGF-β1, TGF-βR1 and TGF-βR2). The expression of Runx-2 was up-regulated. The osteogenic proteins (AP, OSX and OPG) were also overexpressed on osteoblasts cultured on Ti-DoxNPs and Ti-DexNPs. The OPG/RANKL ratio was the highest when DoxNPs were present (75-fold in- crease with respect to the control group). DexNPs also produced a significantly higher OPG/RANKL ratio with respect to the control (20 times higher). Osteoblasts grown on titanium discs were mainly flat and polygonal in shape, with inter-cellular connections. In contrast, osteoblasts cultured on Ti-DoxNPs or Ti-DexNPs were found to be spindle-shaped and had abundant secretions on their surfaces. Significance: DoxNPs and DexNPs were able to stimulate osteoblasts differentiation when applied on titanium surfaces, being considered potential inducers of osteogenic environment when performing regenerative proce- dures around titanium dental implants.Grant PID2020–114694RB-I00 funded by MCIN/AEI 10.13039/501100011033FPU of Ministry of Universities grant FPU20/0045Klockner S.A. for financial support and for providing the titanium disc

Doxycycline-Doped Polymeric Membranes Induced Growth, Differentiation and Expression of Antigenic Phenotype Markers of Osteoblasts

This research was funded by (1) the Ministry of Economy and Competitiveness and European Regional Development Fund [Project MAT2017-85999-P MINECO/AEI/FEDER/UE], (2) University of Granada/Regional Government of Andalusia Research Fund from Spain and European Regional Development Fund (A-BIO-157-UGR-18/FEDER). This research is part of Manuel Toledano-Osorio’s PhD research study.Polymeric membranes are employed in guided bone regeneration (GBR) as physical barriers to facilitate bone in-growth. A bioactive and biomimetic membrane with the ability to participate in the healing and regeneration of the bone is necessary. The aim of the present study was to analyze how novel silicon dioxide composite membranes functionalized with zinc or doxycycline can modulate the osteoblasts' proliferation, differentiation, and expression of selected antigenic markers related to immunomodulation. Nanostructured acrylate-based membranes were developed, blended with silica, and functionalized with zinc or doxycycline. They were subjected to MG63 osteoblast-like cells culturing. Proliferation was assessed by MTT-assay, differentiation by evaluating the alkaline phosphatase activity by a spectrophotometric method and antigenic phenotype was assessed by flow cytometry for selected markers. Mean comparisons were conducted by one-way ANOVA and Tukey tests (p < 0.05). The blending of silica nanoparticles in the tested non-resorbable polymeric scaffold improved the proliferation and differentiation of osteoblasts, but doxycycline doped scaffolds attained the best results. Osteoblasts cultured on doxycycline functionalized membranes presented higher expression of CD54, CD80, CD86, and HLA-DR, indicating a beneficial immunomodulation activity. Doxycycline doped membranes may be a potential candidate for use in GBR procedures in several challenging pathologies, including periodontal disease.European Commission MAT2017-85999-P A-BIO-157-UGR-18/FEDERMinistry of Economy and Competitiveness MAT2017-85999-PUniversity of Granada/Regional Government of Andalusia Research Fund from Spain A-BIO-157-UGR-18/FEDE

Human Fibroblast Gene Expression Modulation Using 940 NM Diode Laser

Low-Level Laser Therapy is used as regenerative therapy in different clinical fields. This is due to its photobiomodulation effect via cell signaling on different cell populations, Including fibroblasts, cells involved in tissue regeneration and healing. The aim was to analyze the effect of 940 nm diode laser on the gene expression of different markers involved in fibroblast growth, differentiation, and migration. Real-time polymerase chain reaction (q-RT-PCR) was used to quantify the expression of fibroblast growth factor (FGF), connective tissue growth factor (CTGF), vascular-endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), TGFβ-receptors (TGFβR1, TGFβR2, and TGFβR3), discoidin-domain receptor-2 (DDR2), matrix metalloproteinase-2 (MMP2), α-actin, fibronectin, decorin, and elastin on human fibroblast, treated with single dose (T1) or two doses (T2) of diode laser at 0.5 Watts and 4 J/cm2. A significant increase in the expression of FGF, TGF-β1, TGFβR1, TGFβR2, α-actin, fibronectin, decorin, DDR2 and MMP2 was observed after both treatments. A decrease was observed in expression of elastin (T1 and T2), and CTGF (T2). These changes underlie the biostimulatory effect of laser on fibroblasts, which translates into an increase in short-term proliferation and in long-term differentiation to myofibroblasts. These data support the therapeutic potential of diode laser for wound repair

Bone Protective Effect of Extra-Virgin Olive Oil Phenolic Compounds by Modulating Osteoblast Gene Expression

The phenolic compounds of extra-virgin olive oil can act at various levels to protect individuals against cardiovascular and neurodegenerative diseases, cancer, and osteoporosis, among others. Polyphenols in extra-virgin olive oil can stimulate the proliferation of osteoblasts, modify their antigen profile, and promote alkaline phosphatase synthesis. The objective of this work was to determine the effect of different extra-virgin olive oil phenolic compounds on the gene expression of osteoblast-related markers. The cells of the MG63 osteoblast line were cultured for 24 h with 10-6 M of the phenolic compounds ferulic acid, caffeic acid, coumaric acid, apigenin, or luteolin. The expression of studied markers was quantified using quantitative real-time polymerase chain reaction (q-RT-PCR). The expression by MG63 osteoblasts of growth and differentiation/maturation markers was modified after 24 h of treatment with 10-6 M of the phenolic compounds under study, most of which increased the gene expression of the transforming growth factor 1 (TGF- 1), TGF- receptor 1,2 and 3 (TGF- R1, TGF- R2, TGF- R3), bone morphogenetic protein 2 and 7 (BMP2, BMP7), run-related transcription factor 2 (RUNX-2), Alkaline phosphatase (ALP), Osteocalcin (OSC), Osterix (OSX), Collagen type I (Col-I) and osteoprotegerin (OPN). The extra-virgin olive oil phenolic compounds may have a beneficial effect on bone by modulating osteoblast physiology, which would support their protective effect against bone pathologies.The work outlined in this article has been partially funded by the Spanish Ministry of Education under FPU fellowship reference FPU15-0563

Effect of olive oil phenolic compounds on osteoblast differentiation

"This is the peer reviewed version of the following article: Melguizo-Rodríguez L., Manzano-Moreno F.J., De Luna-Bertos E., Rivas A., Ramos-Torrecillas J., Ruiz C., García-Martínez O. Effect of olive oil phenolic compounds on osteoblast differentiation. European Journal of Clinical Investigation, 2018; 48(4): 1-6, which has been published in final form at 10.1111/eci.12904. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited."Background: Osteoporosis is a skeletal disorder characterized by compromised bone strength that predisposes individuals to an increased risk of fracture. Previous in vivo and in vitro studies have reported that phenolic compounds present in extra virgin olive oil have a beneficial effect on osteoblasts in terms of increase cell proliferation. The aim of this study was to determine whether phenolic compounds present in olive oil could modify the expression of cell differentiation markers on osteoblasts. Study Design: An in vitro experimental design was peformed using MG-63 osteoblasts cell line. Methods: MG63 cells were exposed to different doses of luteolin, apigenin, or p-coumaric, caffeic, or ferulic acid. Alkaline phosphatase (ALP) was evaluated by spectrophotometry and antigen expression (CD54, CD80, CD86, and HLA-DR) by flow cytometry. Results: At 24 h, treated groups showed an increased ALP and modulated antigen profile, with respect to the non-treated group. Conclusion: These results demonstrate that the phenolic compounds studied induce cell maturation in vitro, increasing ALP synthesis and reducing the expression of antigens involved in immune functions of the osteoblast which would improve bone density.GRUPO BIO-27