Javier Ramos-Torrecillas

Effect and Clinical Implications of the Low-Energy Diode Laser on Bone Cell Proliferation

Laser is a simple, noninvasive technique that has proven useful for treating damaged tissue. However, its effects on bone regeneration and the mechanisms involved are poorly understood. The objective of this study was to evaluate the effects on MG-63 cell proliferation of application of a pulsed diode laser (Ezlase) of 940 nm at low energy levels. After 24 hr of culture, osteoblasts underwent pulsed laser radiation at 0.5, 1, 1.5, and 2 W and fluences of 1–5 J. A control group was not irradiated. After the treatment, cells were incubated for 24 hr, and cell proliferation was analyzed using a spectrophotometric measure of cell respiration (MTT assay). Results were expressed as percentage proliferation versus controls. At 24-hr culture, cell proliferation was increased in laser-treated cells at intensities of 0.5, 1, and 1.5 W/cm2 versus controls; the energy density was positively correlated with cell growth, which reached a peak at 3 J and decreased at higher fluences. The use of pulsed low-level laser with low-energy density range thus appears to exert a biostimulatory effect on bone tissue. Although the data on cell proliferation are robust, in-depth investigation is required into the effect of these irradiation doses on other cell parameters. The present findings demonstrate that laser therapy could be highly useful in tissue regeneration in different clinical settings, including nursing, physical therapy, dentistry, and traumatology.

Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation

In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11–16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18–22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9–13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly.

Discovery of a new binding site on human choline kinase α1: design, synthesis, crystallographic studies, and biological evaluation of asymmetrical bispyridinium derivatives.

Human choline kinase α (CKα) is a validated drug target for the treatment of cancer. In recent years, a large number of CK inhibitors have been synthesized, and one of them is currently being evaluated in Phase I clinical trials as a treatment for solid tumors. Here we have evaluated a new series of asymmetrical biscationic CK inhibitors by means of enzymatic, crystallographic, and antitumor studies. We demonstrate that one of these structures adopts a completely new binding mode not observed before inducing the aperture of an adjacent binding site. This compound shows antiproliferative and apoptotic effects on cancer cells through activation of caspase-3. Therefore, this study not only provides fruitful insights into the design of more efficient compounds that may target different regions in CKα1 but also explains how these compounds induce apoptosis in cancer cells.

Effectiveness of Platelet-Rich Plasma and Hyaluronic Acid for the Treatment and Care of Pressure Ulcers

Platelet-rich growth factor (PRGF) is a natural source of growth factors (GF), while hyaluronic acid (HA) is a biopolymer present in the extracellular matrix of skin, cartilage, bone, and brain, among other tissues. Both are involved in the pathophysiological mechanisms underlying wound healing. The objective of this study was to evaluate the clinical efficacy (as measured by ulcer area) and safety (as measured by signs of infection) of PRGF and PRGF plus HA in the treatment of pressure ulcers (PUs). Patients (N = 100) with 124 Stage II–III PUs were randomized to a control group (n = 25 PUs) for standard care or to case groups for treatment with one (n = 34 PUs) or two (n = 25 PUs) doses of PRGF from their own peripheral blood, or two doses of PRGF plus HA (n = 40 PUs). All ulcers were followed up every 3 days for a 36-day period. At 36 days, a significant reduction in ulcer area (p ≤ .001) was observed in all treatment groups, with a mean reduction of more than 48.0% versus baseline. The greatest mean reduction (80.4% vs. baseline) was obtained with the PRGF plus HA regimen. Complete wound healing was observed in 32.0% of PUs treated with two doses of PRGF (p ≤ .002) and in 37.5% of those treated with two doses of PRGF plus HA (p ≤ .004). There were no signs of infection in any PUs during the 36-day follow-up period. The degree of wound healing was inversely correlated with the consumption of drugs such as statins and with the peripheral blood platelet levels of patients at baseline.

Repercussions of NSAIDS drugs on bone tissue: the osteoblast.

Non-steroidal anti-inflammatory drugs (NSAIDs) can act by modulating the behavior of osteoblasts, including their proliferation, differentiation, adhesion, and migration, but not all NSAIDs have these effects. Our objective was to update the information on this issue in a review of the literature in order to offer guidance on the prescription of the appropriate NSAID(s) to patients requiring bone tissue repair. To review current knowledge of this issue by searching for all relevant publications since 2001 in the MEDLINE, EMBASE and Cochrane Library databases, we used the following descriptors: bone tissue, osteoblast, NSAIDs, Anti-inflammatory drugs. Published studies show that most NSAIDs have an adverse effect on osteoblast growth by cell cycle arrest and apoptosis induction. The effect on differentiation varies according to the drug, dose, and treatment time. Osteoblast adhesion is increased and migration decreased by some NSAIDs, such as indomethacin and diclofenac. The antigenic profile or phagocytic function can also be modulated by NSAIDs. In general, NSAIDs have an adverse effect on bone tissue and given the routine administration of NSAIDs to individuals requiring bone repair, in which the osteoblast has an essential role, this effect on bone should be borne in mind.

The effect of low-level diode laser therapy on early differentiation of osteoblast via BMP-2/TGF-β1 and its receptors.

OBJECTIVES The objective of this study was to determine the effect of LLDL therapy on the gene expression of osteoblast markers of growth and differentiation. MATERIALS AND METHODS The MG-63 cell line was exposed to diode laser (ezLase) of 940 nm at 1-1.5 W and 3-4 J, and gene expressions (Runx-2, alkaline phosphatase [ALP], type I collagen [Col-I], osterix [OSX], osteocalcin [OSC], osteoprotegerin [OPG], bone morphogenetic protein [BMP]-2 and -7, transforming growth factor-β1 [TGF-β1], and TGF-β receptors [TGF-β R1, TGF-β R2; TGF-β R3]) were evaluated by quantitative RT-PCR. RESULTS LLDL treatment stimulated the expression of osteoblast differentiation markers ALP, Col-I, Runx-2, and OSX in relation to the doses applied (P < 0.05), but no changes were detected in OSC, OPG, or BMP-7 at any study dose. This effect may be mediated by TGF-β1 and BMP-2, given that the treatment increased their expression and that of TGF-β receptors R1, R2, and R3 (P < 0.001). CONCLUSION These results suggest that the biostimulatory effect of laser therapy on osteoblasts may be attributable to the release of autocrine factors in response to the irradiation. A clinical trial is warranted to test its therapeutic usefulness in bone tissue regeneration and to define a treatment protocol.

Antiproliferative Activity, Cell Cycle, and Apoptosis Studies of a Series of 6-Substituted 9H-Purin-9-yl-pyridinium Derivatives on a Human Cervical Carcinoma Cell Line

Physicochemical parameters are among the most relevant properties to be evaluated in the early phase of drug development. Lipophilicity is one physicochemical parameter for which early prescreening is available. Correlation analysis between lipophilicity and such biological activities as cytotoxicity, antiproliferative effects, and apoptosis has a significant impact on drug discovery, as it can be used to predict and estimate biological efficacy. Choline kinase (ChoK) is overexpressed in human tumors. Small-molecule inhibitors that can interfere with ChoK activity have proven to be effective antitumor drugs in vitro and in vivo. We recently reported the rational development of a series of nonsymmetrical ChoK inhibitors bearing a 4-substituted pyridinium ring and an adenine moiety connected by linkers of various lengths (Figure 1, compounds 1–8). These structures were biologically evaluated and showed moderate potency as antiproliferative agents against the human hepatoma HepG2 cell line. Considering that lipophilicity can be easily modified and determined by characterizing molecules prepared with different moieties, herein we present a new series of compounds in which the polar amino group at position 6 of the adenine (Figure 1, compounds 1–8, X=NH2) was replaced with a benzylthio substituent (Figure 1, compounds 9–16, X=SCH2Ph) to increase their lipophilicity. The antiproliferative and cytotoxic effects of compounds 1–16 were evaluated against a human cervical carcinoma (HeLa) cell line to study how lipophilicity influences biological activity. Oncogenic transformations lead to cell-cycle aberration and dysregulation of apoptosis. The targeting of cell-cycle and apoptotic pathways has emerged as an attractive approach for the treatment of cancer. In this study we evaluated the effect of compounds 9–16 on cell-cycle progression and apoptosis in HeLa cells. Finally, the most apoptotic compound, 12, was selected for further investigations of downstream apoptotic signaling by analyzing the activation of caspase-3. Synthesis of the target compounds 9–16 was carried out as shown in Scheme 1. 6-Benzylthiopurine (17) was synthesized by a nucleophilic substitution reaction between benzyl bromide and 6-mercaptopurine. The strategy for synthesizing monocationic intermediates 18–25 was based on our previous reported method. Finally, the purine alkylation at N9 was carried out by microwave-assisted synthesis with DMF as a solvent, irradiating at 130 8C for 30 min. Carcinoma of the cervix is considered to be relatively resistant to chemotherapy. Few cytotoxic agents have shown significant activity in advanced and recurrent cervical cancer. Thus, the effects on cell proliferation were investigated in HeLa cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay. Table 1 lists the IC50 values of Figure 1. Structures of monoquaternary pyridinium salts 1–16.

Human fibroblast-like cultures in the presence of platelet-rich plasma as a single growth factor source: clinical implications.

OBJECTIVE: The purpose of this study was to compare the proliferation, morphology, and antigenic expression of human fibroblast–like cells between primary cultures treated with platelet-rich plasma (PRP) or fetal bovine serum (FBS) as the growth factor source. DESIGN: Cells from human gingival tissue samples obtained from healthy volunteers during oral surgery were studied. Isolated cells were cultured in media supplemented with 10% PRP or FBS. Platelet-rich plasma was prepared from the venous blood of each patient. The authors studied short- and long-term cell cultures in the presence of PRP or FBS as the sole growth factor source in order to determine (a) cell growth rate, by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay; (b) cell morphology, by electronic microscopy; and (c) antigenic expression, by flow cytometry and confocal microscopy. RESULTS: In short-term cultures, the cell growth rate was higher with PRP versus FBS treatment. No differences in morphology or expression of vimentin, fibronectin, or &agr;-actin antigens were observed between PRP and FBS cultures. In long-term cultures, PRP and FBS did not significantly differ in cell growth rate but differed in morphology and in the expression of vimentin, fibronectin, and &agr;-actin. CONCLUSION: The PRP enhances cell proliferation over the short term and induces cell differentiation of fibroblast-like cells to myofibroblast-like cells over the long term, suggesting that fibroblast differentiation to myofibroblasts may underlie the action mechanism of PRP in soft tissue regeneration.

Multifunctional capacity and therapeutic potential of lactoferrin

Lactoferrin (LF) is a glycoprotein with high functional versatility that is found in most body fluids. The objective of this study was to gather and update information on the properties attributed to LF. According to this review, LF is a good immunomodulatory agent that acts on both innate and adaptive immune responses. It possesses antimicrobial activity against parasites, fungi, and viruses and also has regenerative properties at tissue level and anti-carcinogenic activity. All of these properties endow LF with major therapeutic potential of which little advantage has been taken to date.

Bisphosphonate Modulation of the Gene Expression of Different Markers Involved in Osteoblast Physiology: Possible Implications in Bisphosphonate-Related Osteonecrosis of the Jaw

The aim of the present study was to elucidate the role of osteoblasts in bisphosphonates-related osteonecrosis of the jaw (BRONJ). The specific objective was to evaluate the effect on osteoblasts of two nitrogen-containing BPs (zoledronate and alendronate) and one non-nitrogen-containing BP (clodronate) by analyzing modulations in their expression of genes essential for osteoblast physiology. Real-time polymerase chain reaction (RT-PCR) was used to study the effects of zoledronate, alendronate, and clodronate at doses of 10-5, 10-7, or 10-9 M on the expression of Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-β1, VEGF, TGF-βR1, TGF-βR2, and TGF-βR3 by primary human osteoblasts (HOBs) and MG-63 osteosarcoma cells. Expression of these markers was found to be dose-dependent, with no substantive differences between these cell lines. In general, results demonstrated a significant increase in TFG-β1, TGF-βR1, TGF-βR2, TGF-βR3, and VEGF expressions and a significant reduction in RUNX-2, Col-1, OSX, OSC, BMP-2, BMP-7, ALP, and RANKL expressions, while OPG expression varied according to the dose and cell line. The results of this in vitro study of HOBS and MG-63 cell lines indicate that low BP doses can significantly affect the expression of genes essential for osteoblast growth and differentiation and of genes involved in regulating osteoblast-osteoclast interaction, possibly by increasing TGF-β1 production. These findings suggest that osteoblasts may play an important role in BRONJ development, without ruling out other factors.