Doxycycline-doped collagen membranes accelerate in vitro osteoblast proliferation and differentiation.

OBJECTIVE: The aim of the study was to evaluate the effect of doxycycline- and dexamethasone-doped collagen membranes on the proliferation and differentiation of osteoblasts. BACKGROUND: Collagen barrier membranes are frequently used to promote bone regeneration and to boost this biological activity their functionalization with antibacterial and immunomodulatory substances has been suggested. METHODS: The design included commercially available collagen membranes doped with doxycycline (Dox-Col-M) or dexamethasone (Dex-Col-M), as well as undoped membranes (Col-M) as controls, which were placed in contact with cultured MG63 osteoblast-like cells (ATCC). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and differentiation by measuring the alkaline phosphatase (ALP) activity using spectrophotometry. Real-time quantitative polymerase chain reaction was used to study the expression of the genes: Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3. Scanning electron microscopy was used to study osteoblast morphology. Data were assessed using one-way analysis of variance or Kruskal-Wallis tests, once their distribution normality was assessed by Kolmogorov-Smirnov tests (p > .05). Bonferroni for multiple comparisons were carried out (p < .05). RESULTS: Osteoblast proliferation was significantly enhanced in the functionalized membranes as follows: (Col-M < Dex-Col-M < Dox-Col-M). ALP activity was significantly higher on cultured osteoblasts on Dox-Col-M. Runx-2, OSX, ALP, OSC, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 were overexpressed, and RANKL was down-regulated in osteoblasts cultured on Dox-Col-M. The osteoblasts cultured in contact with the functionalized membranes demonstrated an elongated spindle-shaped morphology. CONCLUSION: The functionalization of collagen membranes with Dox promoted an increase in the proliferation and differentiation of osteoblasts.

Modulation of Osteogenic Gene Expression by Human Osteoblasts Cultured in the Presence of Bisphenols BPF, BPS, or BPAF.

Bone effects attributed to bisphenols (BPs) include the inhibition of growth and differentiation. This study analyzes the effect of BPA analogs (BPS, BPF, and BPAF) on the gene expression of the osteogenic markers RUNX2, osterix (OSX), bone morphogenetic protein-2 (BMP-2), BMP-7, alkaline phosphatase (ALP), collagen-1 (COL-1), and osteocalcin (OSC). Human osteoblasts were obtained by primary culture from bone chips harvested during routine dental work in healthy volunteers and were treated with BPF, BPS, or BPAF for 24 h at doses of 10-5, 10-6, and 10-7 M. Untreated cells were used as controls. Real-time PCR was used to determine the expression of the osteogenic marker genes RUNX2, OSX, BMP-2, BMP-7, ALP, COL-1, and OSC. The expression of all studied markers was inhibited in the presence of each analog; some markers (COL-1; OSC, BMP2) were inhibited at all three doses and others only at the highest doses (10-5 and 10-6 M). Results obtained for the gene expression of osteogenic markers reveal an adverse effect of BPA analogs (BPF, BPS, and BPAF) on the physiology of human osteoblasts. The impact on ALP, COL-1, and OSC synthesis and therefore on bone matrix formation and mineralization is similar to that observed after exposure to BPA. Further research is warranted to determine the possible contribution of BP exposure to the development of bone diseases such as osteoporosis.

Human adipose tissue-derived mesenchymal stromal cells and their phagocytic capacity.

Mesenchymal stromal cells (MSCs) have evidenced considerable therapeutic potential in numerous clinical fields, especially in tissue regeneration. The immunological characteristics of this cell population include the expression of Toll-like receptors and mannose receptors, among others. The study objective was to determine whether MSCs have phagocytic capacity against different target particles. We isolated and characterized three human adipose tissue MSC (HAT-MSC) lines from three patients and analysed their phagocytic capacity by flow cytometry, using fluorescent latex beads, and by transmission electron microscopy, using Escherichia coli, Staphylococcus aureus and Candida albicans as biological materials and latex beads as non-biological material. The results demonstrate that HAT-MSCs can phagocyte particles of different nature and size. The percentage of phagocytic cells ranged between 33.8% and 56.2% (mean of 44.37% ± 11.253) according to the cell line, and a high phagocytic index was observed. The high phagocytic capacity observed in MSCs, which have known regenerative potential, may offer an advance in the approach to certain local and systemic infections.

Effect of the most common wound antiseptics on human skin fibroblasts.

BACKGROUND: Antiseptics are used for the cleansing of acute or chronic wounds to eliminate micro-organisms from the wound bed. However, they have effects on the skin cells. AIM: To determine the effects of hexetidine, povidone-iodine (PI), undecylenamidopropyl-betaine/polyhexanide (UBP), chlorhexidine, disodium eosin and hydrogen peroxide on human skin fibroblasts. METHODS: CCD-1064Sk cells were treated with hexetidine, PI, UBP, chlorhexidine, disodium eosin or hydrogen peroxide. Spectrophotometry was used to measure cell viability and flow cytometry was used to study apoptosis and necrosis after the treatment. In vitro wound scratch assays were performed to determine the gap closure. RESULTS: All antiseptics significantly reduced the viability of human skin fibroblasts compared with controls. The percentage wound closure was lower with hexetidine, PI and UBP. The scratch assay could not be measured after treatments with chlorhexidine, disodium eosin or hydrogen peroxide, owing to their cytotoxicity. The apoptosis/necrosis experiments evidenced a significant reduction in viable cells compared with controls. An increased percentage of apoptotic cells was observed after treatment with all antiseptics. Compared with controls, the percentage of necrotic cells was significantly increased with all antiseptics except for hexetidine. CONCLUSION: The proliferation, migration and viability of human skin fibroblasts are reduced by treatment with hexetidine, PI, UBP, chlorhexidine, disodium eosin and hydrogen peroxide.

Repercussions of Bisphenol A on the Physiology of Human Osteoblasts.

(1) Background: Bisphenol A (BPA) is an endocrine disruptor that is widely present in the environment and exerts adverse effects on various body tissues. The objective of this study was to determine its repercussions on bone tissue by examining its impact on selected functional parameters of human osteoblasts. (2) Methods: Three human osteoblast lines were treated with BPA at doses of 10-5, 10-6, or 10-7 M. At 24 h post-treatment, a dose-dependent inhibition of cell growth, alkaline phosphatase activity, and mineralization was observed. (4) Results: The expression of CD54 and CD80 antigens was increased at doses of 10-5 and 10-6 M, while the phagocytic capacity and the expression of osteogenic genes (ALP, COL-1, OSC, RUNX2, OSX, BMP-2, and BMP-7) were significantly and dose-dependently reduced in the presence of BPA. (5) Conclusions: According to these findings, BPA exerts adverse effects on osteoblasts by altering their differentiation/maturation and their proliferative and functional capacity, potentially affecting bone health. Given the widespread exposure to this contaminant, further human studies are warranted to determine the long-term risk to bone health posed by BPA.

Antimicrobial properties of olive oil phenolic compounds and their regenerative capacity towards fibroblast cells.

Some micronutrients of vegetable origin are considered potentially useful as wound-healing agents because they can increase fibroblast proliferation and differentiation. THE AIM OF THIS STUDY: was to evaluate the regenerative effects of selected olive oil phenolic compounds on cultured human fibroblasts and explore their antimicrobial properties. MATERIAL AND METHODS: The CCD-1064Sk fibroblast line was treated for 24 h with 10-6M luteolin, apigenin, ferulic, coumaric acid or caffeic acid, evaluating the effects on cell proliferation by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) spectrophotometric assay; the migratory capacity by the scratch assay and determining the expression of Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor- ß1 (TGFß1), Platelet Derived Growth Factor (PDGF), and Collagen Type I (COL-I) genes by real-time polymerase chain reaction. The antimicrobial capacity of the polyphenols was evaluated by the disc diffusion method. RESULTS: All compounds except for ferulic acid significantly stimulated the proliferative capacity of fibroblasts, increasing their migration and their expression of the aforementioned genes. With respect to their antimicrobial properties, treatment with the studied compounds inhibited the growth of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Proteus spp., and Candida Albicans. CONCLUSIONS: The phenolic compounds in olive oil have a biostimulatory effect on the regeneration capacity, differentiation, and migration of fibroblasts and exert major antibacterial activity. According to the present findings, these compounds may have a strong therapeutic effect on wound recovery.

Advantages of Maggot Debridement Therapy for Chronic Wounds: A Bibliographic Review.

GENERAL PURPOSE: To present an overview of the advantages of maggot debridement therapy as a treatment for chronic wounds through the review of several larval properties. TARGET AUDIENCE: This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. LEARNING OBJECTIVES/OUTCOMES: After participating in this educational activity, the participant will be able to:1. Summarize the use, process, and precautions for maggot debridement to treat chronic wounds.2. Synthesize the results of the bibliographic review of the use of maggot debridement to treat chronic wounds. ABSTRACT: Maggot debridement therapy (MDT) is effective for ulcer debridement, achieving it in less time than other therapies. It offers a benefit to healing. However, it is unclear whether maggots reduce treatment time and there is considerable controversy around the treatment's potential antimicrobial action and cost-effectiveness. Nevertheless, it can be effective in preventing amputations and reducing the need for systemic antibiotics. This bibliographic review assesses the advantages of MDT as a treatment for chronic wounds through the review of several larval properties. The review was carried out by consulting biomedical databases including CINAHL, MEDLINE (PubMed), and Scopus, and concludes that MDT is an effective debridement and potential technique to facilitate healing. However, more data is needed on the wound type application frequency and the efficacy of treatment.

Maggot debridement therapy (MDT) is effective for ulcer debridement, achieving it in less time than other therapies. It offers a benefit to healing. However, it is unclear whether maggots reduce treatment time and there is considerable controversy around the treatment's potential antimicrobial action and cost-effectiveness. Nevertheless, it can be effective in preventing amputations and reducing the need for systemic antibiotics. This bibliographic review assesses the advantages of MDT as a treatment for chronic wounds through the review of several larval properties. The review was carried out by consulting biomedical databases including CINAHL, MEDLINE (PubMed), and Scopus, and concludes that MDT is an effective debridement and potential technique to facilitate healing. However, more data is needed on the wound type application frequency and the efficacy of treatment.

Influence of pH on osteoclasts treated with zoledronate and alendronate.

OBJECTIVES: The objectives of this study were to analyze the effect of pH on the growth and activity of osteoclasts treated with different doses of two nitrogen-containing BPs, zoledronate and alendronate. MATERIALS AND METHODS: Murine osteoclasts cultured on dentine disks were treated with zoledronate (50 or 500 nM) or alendronate (500 or 5 µM) at two different pH values (7.4 or 7.0). Osteoclasts were counted with transmitted light microscopy, apoptosis/necrosis was studied with flow cytometry and confocal microscopy, and resorption pit number and depth were calculated using reflected light and scanning electron microscopy. RESULTS: The osteoclast count on dentine disks was significantly (p < 0.001) reduced by zoledronate or alendronate treatment at pH 7.0 in comparison to treatment with the same doses at pH 7.4 and untreated disks (controls). The percentage of apoptotic cells was significantly increased by treatment with 500 nM zoledronate or 5 µM alendronate at pH 7.0 in comparison to the same doses at pH 7.4. The number and depth of resorption pits were significantly lower in disks treated at each BP dose studied than in untreated controls at pH 7.0. CONCLUSIONS: Zoledronate and alendronate at therapeutic doses have an adverse effect on the viability and resorptive activity of osteoclasts when the local medium pH is reduced. CLINICAL RELEVANCE: These findings suggest that periodontal or peri-implant oral cavity infection may be a key trigger of the cascade of events that lead to BRONJ.

Effect of olive oil phenolic compounds on osteoblast differentiation.

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 performed 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 (cluster of differentiation [CD] 54, CD80, CD86 and HLA-DR) by flow cytometry. RESULTS: At 24 hour, treated groups showed an increased ALP and modulated antigen profile, with respect to the nontreated 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.

Effect of Clodronate on Antigenic Profile, Growth, and Differentiation of Osteoblast-Like Cells.

PURPOSE: To evaluate the role of osteoblasts in bisphosphonate-related osteonecrosis of the jaw (BRONJ) by studying the effects of different concentrations of clodronate, a non-nitrogen-containing bisphosphonate, on osteoblast growth, differentiation, and antigenic profile. MATERIALS AND METHODS: Osteoblast-like cells (MG63) were incubated in culture medium with different doses of clodronate. Their proliferative capacity was determined with a spectrophotometric technique (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium assay). Flow cytometry was used to study the antigenic profile. Cell differentiation was evaluated by nodule formation and alkaline phosphatase (ALP) activity was measured by spectrophotometric assay. RESULTS: Clodronate had a significant stimulatory effect on osteoblast-like cell (MG63) proliferation (P < .05). A significant decrease in the expression of CD54, CD80, CD86, and HLA-DR membrane antigens versus controls was observed after 24 hours of treatment with the different clodronate doses assayed (P < .05). A significant decrease (P = .004) in ALP activity was found after 24 hours of treatment with the lowest dose (10(-9) mol/L), and a significant decrease in calcium deposition was found after 15 and 21 days of treatment (P < .05). CONCLUSION: Clodronate increases the proliferation of MG63 osteoblast-like cells and decreases their differentiation capacity, generally at low doses, and modulates the expression of costimulatory molecules associated with immune function. Clodronate exerts its effect on osteoblasts by altering their physiology and impairing their repair capacity, which could be related to the development of BRONJ. However, further research is warranted to elucidate fully the mechanisms by which bisphosphonates can produce this disease.

Nitrogen-containing bisphosphonates modulate the antigenic profile and inhibit the maturation and biomineralization potential of osteoblast-like cells.

OBJECTIVES: The aim was to evaluate the effect of three nitrogen-containing bisphosphonates at different concentrations on osteoblast growth, differentiation, and antigenic profile, using the MG-63 cell line as osteoblast model, in order to determine the role of osteoblasts in bisphosphonate-related osteonecrosis of the jaw (BRONJ). MATERIALS AND METHODS: Osteoblasts were incubated in culture medium with 10(-5), 10(-7), or 10(-9) M of pamidronate, alendronate, or ibandronate. Proliferative capacity of the osteoblasts was determined by spectrophotometry (MTT) at 24 and 48 h of culture. Flow cytometry was used to study antigenic profile (CD54, CD80, CD86, HLA-DR) and phagocytic activity. Cell differentiation was evaluated at 7, 15, and 21 days by the study of nodule formation and alkaline phosphatase activity (ALP) at 24 h by spectrophotometric assay. RESULTS: Pamidronate, alendronate, and ibandronate each exerted a significant stimulatory effect on MG63 proliferation that depended on the dose and treatment duration (p < 0.05). In general, a significantly decreased expression of CD54, CD80, and HLA-DR membrane antigens was observed after 24 h of treatment with each nitrogen-containing bisphosphonate (p < 0.05), but there was no significant difference in phagocytic activity versus controls. A decrease in ALP activity was observed after 24 h of treatment and a decrease in calcium deposition after 15 and 21 days (p < 0.05). CONCLUSION: Nitrogen-containing bisphosphonates can increase the proliferation of MG-63 osteoblast-like cells, modulate their expression of co-stimulatory molecules associated with immune function, and decrease their differentiation capacity, generally at low doses. CLINICAL RELEVANCE: These findings suggest that low doses of nitrogen-containing bisphosphonates exert their effect on osteoblasts by altering their physiology, which would explain the disruption of their repair capacity and may be directly related to the development of BRONJ.

The effects of low-level diode laser irradiation on differentiation, antigenic profile, and phagocytic capacity of osteoblast-like cells (MG-63).

Previous in vivo and in vitro studies have reported that low-level diode laser therapy induces a biostimulatory effect, such as cell proliferation. The aim of the present study was to evaluate whether the laser irradiation of osteoblast-like cells (MG-63) can modify alkaline phosphatase activity (ALP), antigenic profile, and phagocytic capacity. The MG-63 cell line was exposed to diode laser (ezLase) of 940 nm at 1-1.5 W/cm(2) and 3-4 J. ALP was evaluated by a spectrophotometric technique and antigenic expression analysis (CD 54, CD80, CD86, HLA-DR), and phagocytic activity was analyzed by flow cytometry. At 24 h, the treated groups showed an increased ALP, and the highest increase versus controls (P = 0.002) was at the dose of 1 W/cm(2) and 3 J; this modulation of the antigenic profile translated into a reduced expression of CD54, CD86, and HLA-DR and a slightly decreased phagocytic capacity with respect to the nonirradiated control group at the different intensities and fluencies assayed. These results demonstrate that laser therapy can exert a biostimulatory effect on osteoblastic cells at different levels, which may be clinically useful in the regeneration of bone tissue.

The effect of olive oil on osteoporosis prevention.

UNLABELLED: The incidence of osteoporosis and associated fractures is found to be lower in countries where the Mediterranean diet is predominant. These observations might be mediated by the active constituents of olive oil and especially phenolic compounds. OBJECTIVE: To review current knowledge by searching for all relevant publications since 2001 in the MEDLINE, EMBASE and Cochrane Library databases, using the descriptors: Mediterranean diet, virgin olive oil, phenols, bone, osteoblast and osteoporosis. RESULTS AND CONCLUSIONS: Published evidence suggests that olive oil phenols can be beneficial by preventing the loss of bone mass. It has been demonstrated that they can modulate the proliferative capacity and cell maturation of osteoblasts by increasing alkaline phosphatase activity and depositing calcium ions in the extracellular matrix. Further research on this issue is warranted, given the prevalence of osteoporosis and the few data available on the action of olive oil on bone.

Effect of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast physiology.

Background/purpose: Amoxicillin and clindamycin are the most effective decontaminants for intraoral bone grafts before their application in bone regeneration without cytotoxic effects on osteoblasts, but their effects on the gene expression of markers involved in osteoblast growth and differentiation remain unclear. The study objective was to determine the effects of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast growth and differentiation. Materials and methods: Real-time polymerase chain reaction (RT-PCR) was performed to explore the effect of 150 µg/mL clindamycin or 400 µg/mL amoxicillin on the gene expression by primary human osteoblasts (HOBs) of runt-related transcription factor 2 (Runx-2), osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OSC), osteoprotegerin (OPG), receptor activator for nuclear factor κ B ligand (RANKL), type I collagen (Col-I), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), TGF-ß1 and TGF-ß receptors (TGF-ßR1, TGF-ßR2, and TGF-ßR3), and vascular endothelial growth factor (VEGF). Results: Treatment with 150 µg/mL clindamycin significantly increased the gene expression of TFG-ß1, TGF-ßR1, TGF-ßR2, TGF-ßR3, RUNX-2, Col-1, OSX, OSC, BMP-2, BMP-7, ALP, VEGF, and RANKL by HOBs. Treatment with 400 µg/mL amoxicillin significantly increased the gene expression of TGF-ß R1, Col-I, OSC, RANKL, and OPG alone. Conclusion: These findings suggest that 150 µg/mL clindamycin is the decontaminant of choice to treat intraoral bone grafts before their application in bone regeneration. The osteogenic and antibacterial properties of clindamycin can favor and accelerate the integration of bone grafts in the oral cavity.

NP-12 peptide functionalized nanoparticles counteract the effect of bacterial lipopolysaccharide on cultured osteoblasts.

OBJECTIVE: To evaluate whether nanoparticles (NPs) functionalized with Tideglusib (TDg, NP-12), and deposited on titanium surfaces, would counteract the effect of bacterial lipopolysaccharide (LPS) on osteoblasts. METHODS: Experimental groups were: (a) Titanium discs (TiD), (b) TiD covered with undoped NPs (Un-NPs) and (c) TiD covered with TDg-doped NPs (TDg-NPs). Human primary osteoblasts were cultured onto these discs, in the presence or absence of bacterial LPS. Cell proliferation was assessed by MTT-assay and differentiation by measuring the alkaline phosphatase activity. Mineral nodule formation was assessed by the alizarin red test. Real-time quantitative polymerase chain reaction was used to study the expression of Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 genes. Osteoblasts morphology was studied by Scanning Electron Microscopy. One-way ANOVA or Kruskal-Wallis and Bonferroni multiple comparisons tests were carried out (p < 0.05). RESULTS: TDg-NPs enhanced osteoblasts proliferation. Similarly, this group increased ALP production and mineral nodules formation. TDg-NPs on titanium discs resulted in overexpression of the proliferative genes, OSC and OSX, regardless of LPS activity. In the absence of LPS, TDg-NPs up-regulated Runx2, COL-I, ALP, BMP2 and BMP7 genes. OPG/RANKL gene ratios were increased about 2500 and 4,000-fold by TDg-NPs, when LPS was added or not, respectively. In contact with the TDg-NPs osteoblasts demonstrated an elongated spindle-shaped morphology with extracellular matrix production. SIGNIFICANCE: TDg-NPs on titanium discs counteracted the detrimental effect of LPS by preventing the decrease on osteoblasts proliferation and mineralization, and produced an overexpression of proliferative and bone-promoting genes on human primary osteoblasts.

Extra Virgin Olive Oil Phenolic Compounds Modulate the Gene Expression of Biomarkers Involved in Fibroblast Proliferation and Differentiation.

Extra virgin olive oil phenolic compounds have been identified as possible biostimulant agents against different pathological processes, including alterations in healing processes. However, there is little evidence on the molecular mechanisms involved in this process. The aim was to analyse the effect of hydroxytyrosol, tyrosol, and oleocanthal on fibroblast gene expression. PCR was used to determine the expression of different differentiation markers, extracellular matrix elements, and growth factors in cultured human fibroblasts CCD-1064Sk treated with different doses of hydroxytyrosol (10-5 M and 10-6 M), tyrosol (10-5 M and 10-6 M), and oleocanthal (10-6 M and 10-7 M). After 24 h of hydroxytyrosol treatment, increased expression of connective tissue growth factor, fibroblast growth factor (FGF), platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor ß1 (TGF-ß1), and their receptors was observed. Tyrosol and olecanthal modulated the expression of FGF and TGFßR1. All phytochemicals tested modified the expression of differentiation markers and extracellular matrix elements, increasing gene expression of actin, fibronectin, decorin, collagen I, and III. Phenolic compounds present in extra virgin olive could have a beneficial effect on tissue regeneration by modulating fibroblast physiology.

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 conducted 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-expression of the main osteogenic proliferative genes (TGF-ß1, TGF-ßR1 and TGF-ßR2). 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 increase 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 procedures around titanium dental implants.

Biomimetic Collagen Membranes as Drug Carriers of Geranylgeraniol to Counteract the Effect of Zoledronate.

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 µM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-ß1 and TGF-ß receptors (TGF-ßR1, TGF-ßR2, and TGF-ßR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal-Wallis and post hoc Bonferroni tests were applied (p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 µM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control (p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group (p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts' markers.

Effect of ibuprofen on proliferation, differentiation, antigenic expression, and phagocytic capacity of osteoblasts.

Ibuprofen is a nonselective nonsteroidal antiinflammatory drug commonly prescribed for acute postsurgical and posttraumatic pain. However, little known is about the effect of this drug on osteoblasts. In this study, we aimed to investigate the effect of ibuprofen on cell proliferation, differentiation, antigenic profile, and phagocytic activity, in a human MG-63 osteosarcoma cell line, as a model of osteoblasts. Flow cytometry was used to study proliferation, antigenic profile, and phagocytic activity, and radioimmunoassay was used to determine osteocalcin synthesis as a cell differentiation marker. Our results showed that therapeutic doses of ibuprofen (5 and 25 µM) did not modify cell proliferation and osteocalcin synthesis in the MG-63 cellular line. However, treatment with a higher dose (25 µM) increased the expression of antigens CD21, CD44, CD80, CD86, and HLA-DR and decreased phagocytic activity. The results indicate that a therapeutic dose of ibuprofen has no adverse effects on growth of the osteoblast-like cells. Treatment with ibuprofen alone may produce some cell activation, which would explain the increase in expression of membrane markers and decrease in phagocytic capacity.

Effect of platelet-rich plasma on growth and antigenic profile of human osteoblasts and its clinical impact.

PURPOSE: In recent years, there has been widespread clinical use of platelet-rich plasma (PRP) to facilitate the regeneration of different tissues. However, few data are available on the effect of PRP on parameters other than cell growth. The aim of the present study was to evaluate the effect of PRP on the cell cycle, antigenic profile, and proliferation of primary cultured human osteoblasts. MATERIALS AND METHODS: The cells in the present study were derived from human bone sections obtained from healthy volunteers during third molar surgery. PRP was prepared from human venous blood and used to culture the cell line obtained from the same patient. Flow cytometry was used to study the cell cycle, antigenic profile, and proliferation. RESULTS: The treatment of osteoblasts with PRP modified the expression of CD54, CD80, CD86, and HLA-DR antigens. PRP treatment increased cell proliferation in the short term, but the cell proliferation capacity diminished in the long term, perhaps owing to cell exhaustion. No change in the cell cycle profile was observed in the PRP-cultured cells. CONCLUSIONS: These results suggest that PRP treatment accelerates bone neoformation with no cell cycle changes that might carry a risk of malignant transformation.