Effect of Low-Level Laser Irradiation (810 nm) on the Proliferation and Differentiation of Osteoblast-Like Cells Cultured on SLA Titanium Discs Exposed to a Peri-implantitis Environment.

Introduction: Elimination of inflammation and re-osseointegration are the major objectives of peri-implantitis therapy. Existing data, however, do not support any decontamination approach. Thus, the present in vitro study aims to assess whether the air-debriding decontamination method with erythritol powder restores the biocompatibility of infected titanium discs and to investigate the potent biomodulatory ability of diode laser (810 nm) irradiation to promote cell proliferation and differentiation of premature osteoblast-like cells (MG63) towards osteocytes. Methods: The experimental groups consisted of cells seeded on titanium discs exposed or not in a peri-implantitis environment with or without biomodulation. Infected discs were cleaned with airflow with erythritol powder. Cell cultures seeded on tricalcium phosphate (TCP) surfaces with or without biomodulation with a laser (810 nm) were used as controls. The study evaluated cell viability, proliferation, adhesion (SEM) at 24, 48 and 72 hours, and surface roughness changes (profilometry), as well as the effects of low-level laser therapy (LLLT) on ALP, OSC, TGF-b1, Runx2, and BMP-7 expression in MG63 cells' genetic profile on days 7, 14, and 21. Results: The MTT assay as well as the FDA/PI method revealed that cell proliferation did not show significant differences between sterile and decontaminated discs at any timepoint. SEM photographs on day 7 showed that osteoblast-like cells adhered to both sterile and disinfected surfaces, while surface roughness did not change based on amplitude parameters. The combination of airflow and LLLT revealed a biomodulated effect on the differentiation of osteoblast-like cells with regard to the impact of laser irradiation on the genetic profile of the MG63 cells. Conclusion: In all groups tested, osteoblast-like cells were able to colonize, proliferate, and differentiate, suggesting a restoration of biocompatibility of infected discs using airflow. Furthermore, photomodulation may promote the differentiation of osteoblast-like cells cultured on both sterile and disinfected titanium surfaces.

Free titanium particles and P. gingivalis lipopolysaccharide create a potentially synergistical effect in a periimplantitis model.

OBJECTIVE: Our aim was to examine the effect of titanium particles and lipopolysaccharide (LPS) from P. gingivalis on the inflammatory profile expression of human gingival fibroblasts (hGFs), cultured on rough titanium discs, in an in vitro peri-implantitis simulation. DESIGN: Human gingival fibroblasts cultured on SLA and TCP surfaces were challenged with LPS, titanium particles or both. At 24, 48 and 72 h after treatment, MTT assay was performed to assess cell proliferation. FDA/PI staining was performed for the same time periods, in order to evaluate cell viability/apoptosis. At 5 and 7 days after the treatment, qPCR was performed to assess gene expressions of IL-6, IL-8 and COL1A1, as well as SEM on titanium discs. RESULTS: All groups presented a significant increase of their population between the time periods of examination. Regarding the interleukin gene expression, the combination of LPS and particles significantly increased the levels of Interleukin-8. Treatment with LPS and particles also induced a significant increase of Interleukin-6 and collagen. FDA/PI microscopy has revealed several apoptotic cells in the treatment groups. SEM micrographs have shown the difficulty of hGFs to adhere on rough surfaces. CONCLUSIONS: The combination of titanium particles and LPS significantly upregulated the expression of IL-6, IL-8 and Col-1a. It appears that particles may arouse similar reactions to the endotoxin, while synergistically intensifying it.

Dosage Effects of an 810 nm Diode Laser on the Proliferation and Growth Factor Expression of Human Gingival Fibroblasts.

Introduction: A substantial amount of evidence supports the positive effect of photobiomodulation on the proliferation and differentiation of various cell types. Several laser wavelengths have been used for wound healing improvement, and their actual outcome depends on the settings utilized during irradiation. However, the heterogeneous wavelengths and laser settings applied in the existing literature make it difficult to draw solid conclusions and comparison of different studies. The aim of the present study is to evaluate and compare the effects of various doses of laser energy, provided by an 810 nm diode, on human gingival fibroblasts in terms of proliferation and expression of growth factors with a pivotal role in wound healing. Methods: Human gingival fibroblasts were cultured on plastic tissue culture and irradiated with 2, 4, 6 or 12 J/cm2. The effects of the low-level laser therapy (LLLT) using an 810 nm diode laser on growth factor expression (EGF, TGF and VEGF) were evaluated by qPCR at 72 hours and 7 days after irradiation. Cell proliferation was evaluated at 24, 48 and 72 hours after LLLT using MTT assay. Results: Energy density of 12 J/cm2 provoked irradiated gingival fibroblasts to demonstrate significantly higher proliferation as well as higher gene expression of Col1, VEGF and EGF. LLLT positive effects were obvious up to 7 days post-irradiation. Conclusion: LLLT with 810 nm presents beneficial effects on proliferation, collagen production and growth factor expression in human gingival fibroblast cells. The application of 12 J/cm2 can be suggested as the optimal energy density for the enhancement of the wound healing process.

Low Level Laser Therapy With an 810-nm Diode Laser Affects the Proliferation and Differentiation of Premature Osteoblasts and Human Gingival Fibroblasts In Vitro.

Introduction: Photomodulation is a promising strategy for optimizing tissue healing, but its photomodulatory effects on the synergistic cellular metabolism of gingival and bony tissues remain largely unknown. The aim of the present study was to evaluate the photomodulatory effects of a diode laser (810 nm) on osteoblasts, HGFs and their co-cultures in vitro. Methods: Primary cultures of HGFs, cultures of immature osteoblastic cells (MG63) and their co-cultures were irradiated with a diode laser (810 nm), 15 J/cm2. Cell cultures were examined for cellular proliferation (MTT assay), viability (FDA/PI staining) after 24, 48 and 72 hours and cell differentiation (qPCR of collagen type 1a - COL1a and alkaline phosphatase expressions - ALP) after 7 days. Results: Photomodulation with an 810-nm diode laser increased cell proliferation at all time points. COL1a gene expression increased both in HGF and co-cultures. ALP expression was up-regulated in osteoblastic cultures, but co-cultures with fibroblasts negated this response. Conclusion: The 810-nm diode laser positively affected cell proliferation and viability in all experimental groups. The statistically significant increased COL1a gene expression at 7 days after irradiation both in the irradiated HGF and co-cultures suggests that low-level laser therapy (LLLT) stimulated extracellular matrix (ECM) formation signaling in both cell types.

Immunomodulatory effects of Nd:YAG (1064 nm) and diode laser (810 nm) wavelengths to LPS-challenged human gingival fibroblasts.

OBJECTIVE: Human gingival fibroblasts (hGFs) are involved in inflammatory responses to bacteria by recognizing pathogen-associated molecular patterns. In search of host modulation strategies to increase LPS tolerance, Low level laser therapy (LLLT) has been suggested as an alternative treatment that reduces periodontal tissue inflammation. In this study, we investigate whether 810 nm (diode) and 1064 nm (Nd:YAG) laser wavelengths, modulate pro-inflammatory responses to LPS challenges in hGFs. DESIGN: Primary hGFs were challenged with Porphyromonas gingivalis LPS and irradiated with either Diode (810 nm) or with Nd:YAG (1064 nm) lasers. Cell cultures were examined for cell proliferation by MTT assay and IL-6 and IL-8 expression by qPCR at 24, 48 and 72 h. IL-6 and IL-8 protein levels were detected via ELISA. RESULTS: Naïve hGF populations irradiated with both Diode 810 nm and Nd:YAG 1064 nm lasers demonstrated cellular proliferation (p < 0.05), but LLLT did not affect cellular viability in LPS-challenged cells. IL-6 and IL-8 gene expression levels revealed significant anti-inflammatory effects of irradiation with both examined wavelengths on hGFs challenged with P. gingivalis LPS. Protein levels of these cytokines were increased by LPS challenge. Treatment with LLLT inhibited this increase for both wavelengths evaluated in the study at a statistically significant level particularly for the first 48 h. CONCLUSIONS: The present study demonstrates a modulatory effect of LLLT using both 810 nm diode and Nd:YAG 1064 nm lasers in gingival fibroblasts by decreasing the production of IL-6, IL-8 in response to LPS.

Mechanical Enhancement of Cytocompatible 3D Scaffolds, Consisting of Hydroxyapatite Nanocrystals and Natural Biomolecules, Through Physical Cross-Linking.

Bioinspired scaffolds mimicking natural bone-tissue properties holds great promise in tissue engineering applications towards bone regeneration. Within this work, a way to reinforce mechanical behavior of bioinspired bone scaffolds was examined by applying a physical crosslinking method. Scaffolds consisted of hydroxyapatite nanocrystals, biomimetically synthesized in the presence of collagen and l-arginine. Scaffolds were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), microcomputed tomography, and nanoindentation. Results revealed scaffolds with bone-like nanostructure and composition, thus an inherent enhanced cytocompatibility. Evaluation of porosity proved the development of interconnected porous network with bimodal pore size distribution. Mechanical reinforcement was achieved through physical crosslinking with riboflavin irradiation, and nanoindentation tests indicated that within the experimental conditions of 45% humidity and 37 °C, photo-crosslinking led to an increase in the scaffold's mechanical properties. Elastic modulus and hardness were augmented, and specifically elastic modulus values were doubled, approaching equivalent values of trabecular bone. Cytocompatibility of the scaffolds was assessed using MG63 human osteosarcoma cells. Cell viability was evaluated by double staining and MTT assay, while attachment and morphology were investigated by SEM. The results suggested that scaffolds provided a cell friendly environment with high levels of viability, thus supporting cell attachment, spreading and proliferation.

Osteostimulative calcium phosphosilicate biomaterials partially restore the cytocompatibility of decontaminated titanium surfaces in a peri-implantitis model.

Bacterial peri-implant biofilms, and the chemotherapeutics for their removal alter titanium surface cytocompatibility. In this study we aimed to assess the adjunctive use of an osteostimulative biomaterial utilizing a peri-implantitis model under the hypothesis that it will increase cell migration towards treated titanium surfaces. Acid-etched titanium surfaces were inoculated with a multi-species biofilm model and treated with 1.5% NaOCl in a previously characterized in vitro peri-implantitis model. Cell migration of MG63 cells towards the treated titanium surface (CTRL) was significantly reduced following inoculation with biofilm and chemotherapeutic treatment as compared to sterile controls. Addition of a tricalcium phosphate biomaterial (TCP) as a control for Ca+2 had a small non-significant effect, while BG significantly increased MG63 chemotaxis to titanium to levels comparable to sterile (STE). Similarly, cell viability at 5 days was increased in BG and TCP as compared to CTRL. SEM imaging confirmed the improved cytocompatibility of BG and TCP surfaces as compared to CTRL. Osteostimulative BG exhibited a strong chemotactic effect to osteoblasts, which was stronger than what was expected due to the chemotactic effect of Ca+2 alone (TCP). In addition, substantially increased cell attachment and viability was found on treated implant surfaces as compared to CTRL. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2645-2652, 2018.

Nd:YAG laser radiation (1.064 nm) accelerates differentiation of osteoblasts to osteocytes on smooth and rough titanium surfaces in vitro.

BACKGROUND: A number of studies revealed beneficial effects of low-level laser therapy (LLLT) regarding cell proliferation and differentiation. AIM: To investigate the effect of Nd:YAG (1.064 nm) laser radiation in the proliferation and differentiation potential of MG-63 osteoblast-like cells. Additionally, the effects of the surface configurations were to be evaluated. MATERIAL AND METHODS: MG-63 osteoblast cells were cultured on different surfaces: plastic tissue culture, smooth (polished) titanium-PT and rough titanium-SLA. The effects of both titanium surfaces and low-level laser therapy (LLLT) on cell adhesion were evaluated by the gene expression of molecules involved in cell proliferation and differentiation. In addition, scanning electron microscopy (SEM) and MTT proliferation assays were used to examine cell morphology and proliferation, respectively. RESULTS: Compared to smooth (PT) surfaces, SLA surfaces favoured MG-63 cell differentiation. Following the application of Nd:YAG laser irradiation, cells yielded statistically significantly improved differentiation on both smooth and SLA surfaces compared with non-irradiated surfaces. CONCLUSIONS: The findings of this present study suggest that both surface morphology and Nd:YAG laser irradiation influence the proliferation and differentiation potential of MG-63 cells.

Effect of Nd:YAG Low Level Laser Therapy on Human Gingival Fibroblasts.

Aim. To evaluate the effect of Low Level Laser Therapy (LLLT) on human gingival fibroblasts in terms of proliferation and growth factors' secretion (EGF, bFGF, and VEGF). Materials and Methods. Primary cultures of keratinized mucosa fibroblasts were irradiated by a Nd:YAG laser 1064 nm with the following energy densities: 2.6 J/cm(2), 5.3 J/cm(2), 7.9 J/cm(2), and 15.8 J/cm(2). Controls were not irradiated. Cultures were examined for cell proliferation and growth factors' secretion after 24, 48, and 72 hours. All experimental procedures were performed in duplicate. Data were analyzed by Student's t-test (p < 0.05). Results. All laser-irradiation doses applied promoted a higher cell proliferation at 48 hours in a dose-response relationship compared to controls. This difference reached statistical significance for the cultures receiving 15.8 J/cm(2) (p = 0.03). Regarding EGF, all laser irradiation doses applied promoted a higher secretion at 48 hours in a reverse dose-response pattern compared to controls. This difference reached statistical significance for the cultures receiving 2.6 J/cm(2) (p = 0.04). EGF levels at the other time points, bFGF, and VEGF showed a random variation between the groups. Conclusion. Within the limits of this study, LLLT (Nd:YAG) may induce gingival fibroblasts' proliferation and upregulate the secretion of EGF. Further studies are needed to confirm these results.

Collagen I membranes for tendon repair: effect of collagen fiber orientation on cell behavior.

Tendons have poor spontaneous regenerative capabilities, and complete regeneration is never achieved despite intensive remodeling. In this in vitro study, we characterized two multilamellar collagen I membranes differing in the arrangement of collagen fiber deposition (oriented vs. nonoriented) and compared their mechanical properties. Human dermal fibroblasts and tenocytes were seeded on the two membranes to evaluate the effect of fiber orientation on cell viability and cytoskeletal organization. Results demonstrate that the multilamellar collagen I membrane with oriented fibers has the better mechanical properties and affords optimum cell proliferation and adhesion. Its fiber arrangement provides an instructive pattern for cell growth and may serve to guide the alignment of cells migrating from the ends of a crushed or frayed tendon to obtain a strong, correctly structured tendon, thus providing a viable clinical option for tendon repair.