Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha (HIF-1α), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxiccultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Effect of low-level laser therapy on bisphosphonate-treated osteoblasts / 대한악안면성형재건외과학회지

BACKGROUND: This study investigates the effect of alendronate-treated osteoblasts, as well as the effect of low-level laser therapy (LLLT) on the alendronate-treated osteoblasts. Bisphosphonate decreases the osteoblastic activity. Various treatment modalities are used to enhance the bisphosphonate-treated osteoblasts; however, there were no cell culture studies conducted using a low-level laser. METHODS: Human fetal osteoblastic (hFOB 1.19) cells were treated with 50 μM alendronate. Then, they were irradiated with a 1.2 J/cm² low-level Ga-Al-As laser (λ = 808 ± 3 nm, 80 mW, and 80 mA; spot size, 1 cm²; NDLux, Seoul, Korea). The cell survivability was measured with the MTT assay. The three cytokines of osteoblasts, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF) were analyzed. RESULTS: In the cells treated with alendronate at concentrations of 50 μM and higher, cell survivability significantly decreased after 48 h (p < 0.05). After the applications of low-level laser on alendronate-treated cells, cell survivability significantly increased at 72 h (p < 0.05). The expressions of OPG, RANKL, and M-CSF have decreased via the alendronate. The RANKL and M-CSF expressions have increased, but the OPG was not significantly affected by the LLLT. CONCLUSIONS: The LLLT does not affect the OPG expression in the hFOB cell line, but it may increase the RANKL and M-CSF expressions, thereby resulting in positive effects on osteoclastogenesis and bone remodeling.

Low-level laser therapy affects osseointegration in titanium implants: resonance frequency, removal torque, and histomorphometric analysis in rabbits

OBJECTIVES: The purpose of this study was to investigate the effects of low-level laser therapy (LLLT) with a diode gallium-aluminum-arsenide (Ga-Al-As) low-level laser device on the healing and attachment of titanium implants in bone. MATERIALS AND METHODS: Thirteen New Zealand white male rabbits weighing 3.0+/-0.5 kg were used for this study. Dental titanium implants (3.75 mm in diameter and 8.5 mm in length, US II RBM plus fixture; Osstem, Seoul, Korea) were implanted into both femurs of each rabbit. The rabbits were randomly divided into a LLLT group and a control group. The LLLT was initiated immediately after surgery and then repeated daily for 7 consecutive days in the LLLT group. Six weeks and 12 weeks after implantation, we evaluated and compared the osseointegration of the LLLT group and control group, using histomorphometric analysis, removal torque testing, and resonance frequency analysis (RFA). The results were statistically significant when the level of probability was 0.05 or less based on a non-parametric Mann-Whitney U-test. RESULTS: The implant survival rate was about 96%. Histologically and histomorphometrically, we observed that the titanium implants were more strongly attached in LLLT group than in control group. However, there was no significant difference between the LLLT group and control group in removal torque or RFA. CONCLUSION: Histologically, LLLT might promote cell-level osseointegration of titanium implants, but there was no statistically significant effects.

Biophysical therapy and biostimulation in unfavorable bony circumstances: adjunctive therapies for osseointegration

Dental implants using titanium have greatly advanced through the improvement of designs and surface treatments. Nonetheless, the anatomical limits and physiological changes of the patient are still regarded as obstacles in increasing the success rate of implants further, even with the enhancement of implant products. So there have been many efforts to overcome these limits. The intrinsic potential for bone regeneration can be stimulated through adjuvant treatments with the continuous improvement of implant properties, and this can play an important role in achieving optimum osseointegration toward peripheral bone tissue and securing ultimate long-term implant stability in standard surgical procedures. For this purpose, various chemical, biological, or biophysical measures were developed such as bone grafts, materials, pharmacological agents, growth factors, and bone formation proteins. The biophysical stimulation of bone union includes non-invasive and safe methods. In the beginning, it was developed as a method to enhance the healing of fractures, but later evolved into Pulsed Electromagnetic Field, Low-Intensity Pulsed Ultrasound, and Low-Level Laser Therapy. Their beneficial effects were confirmed in many studies. This study sought to examine bone-implant union and its latest trend as well as the biophysical stimulation method to enhance the union. In particular, this study suggested the enhancement of the function of cells and tissues under a disadvantageous bone metabolism environment through such adjunctive stimulation. This study is expected to serve as a treatment guideline for implant-bone union under unfavorable circumstances caused by systemic diseases hampering bone metabolism or the host environment.

Effect of calcium and vitamin D supplementation on bone formation around titanium implant in osteoporosis-induced rats

PURPOSE: The purpose of this study was to observe the effect of calcium and vitamin D to the titanium implant osseointegration in the osteoporosis-induced animal model. MATERIAL AND METHOD: Thirty-two rats, 10 weeks of age, were divided into two groups: experimental group was ingested additional calcium and vitamin D, and a control group was not. Titanium screw implant(diameter, 2.0 mm; length, 3.5 mm; pitch-height 0.4 mm) were placed into tibia of 32 rats, 16 in the control group and 16 in the experimental group. The rats were sacrificed at 1, 2, 4 and 8 weeks after implantation for histopathologic examination, histomorphometric analysis and immunohistochemistry with fibronectin and collagen type I antibody. RESULT: In histopathological findings, newly formed bone was seen at 2 weeks and became lamellar bone at 4 weeks, and mature trabecullar bone was seen at 8 weeks in experimental group. In control group, thickness of regenerated bone increased till 4 weeks gradually and trabecullar bone was seen at 8 weeks. In histomorphometric analysis, marrow bone density increased significantly in experimental group compared to control group. Fibronectin immunoreactivity was strong at 2 weeks in experimental group and reduced after 4 weeks gradually. But it was maintained continuously from 2 to 8 weeks in control group. Collagen type I immunoreactivity was very strong from 2 to 4 week in experimental group. And the amount of Collagen type I expression was more abundant in experimental group. CONCLUSION: The results of this study suggest that calcium and vitamin D supplementation promote bone healing around titanium implants in osteoporosis induced animals.

Effect of Calcium and Vitamin D Supplementation on Bone Formation Around Titanium Implant

The purpose of this study was to observe the effect of calcium and vitamin D to the titanium implant osseointegration in animal model. 32 rats, 10 weeks of age, were divided into two group: additional calcium and vitamin D supplementation group and a control group. Titanium screw implant(diameter, 2.0mm; length, 3.5mm; pitch-height 0.4 mm) were placed into tibia of 32 rats, 16 in the control group and 16 in the experimental group. The rats were sacrificed at different time interval(1, 2, 4, and 8 weeks after implantation) for histopathologic observation, histomorphometric analysis and immunohistochemistry with osteocalcin and osteopontin antibody. Histopathologically findings, newly formed bone was seen at 1 weeks and became lamellar bone at 2 weeks, and mature trabecullar bone was seen at 4 weeks experimental group. In control group, thickness of regenerated bone increased till 4 weeks gradually and trabecullar bone was seen at 8 weeks. By histomorphometric analysis, bone marrow density was increased significantly at 1 and 2 weeks in experimental group compared to control group. Osteocalcin immunoreactivity was strong at 1 week experimental group and reduced after 4 weeks gradually. But it was continuously weakly from 1 to 4 weeks in control group. Osteopontin immunoreactivity was very strong in newly formed bone from 2 to 8 weeks experimental group. And the amount of osteopontin expression was more abundant in experimental group. The results of this study suggest that calcium and vitamin D supplementation promotes bone healing around dental implants.