Diabetic patients tend to exhibit delayed bone formation and osteoblast differentiation, which results in osteopenia. Recently, numerous clinical reports suggest that 635-nm light irradiation improves bone regeneration and wound healing, and reduces pain in patients suffering from diabetes. The purpose of the present study was to test the hypothesis that 635-nm irradiation can influence bone formation by MC3T3-E1 osteoblasts cultured on high concentrations of glucose(25mmol/L D-glucose) in the presence or absence of phorbol 12-myristate 13-acetate(PMA), and to establish an in vitro pathological model of bone formation. The effect of 635-nm irradiation on bone formation was investigated using Alizarin Red S staining, and alkaline phosphatase enzyme activ ity and calcium deposition assays. In addition, gene expression of the o steogenic markers BMP-2, osterix and osteocalcin were assayed by RT-PCR. Calcium deposition by MC3T3-E1 cells was reduced in the presence of high concentrations of glucose or by PMA supplementation. However, 635-nm irradiation led to an increase in calcium deposition by MC3T3 cells, followed by increased bone mineralization. mRNA expression of BMP-2 and osterix at an early stage and of osteocalcin at a late stage was significantly upregulated by 635-nm irradiation in MC3T3-E1 cells supplemented with high concentrations of glucose. Irradiation at 635 nm increases bone mineralization in MC3T3-E1 cells cultured in vitro on high concentrations of glucose and alters osteogenic gene expression, which accelerates bone formation in hyperglycemic conditions.

I. INTRODUCTION
 II. MATERIALS AND METHODS
  1. Cell culture and chemicals
  2. Light source and irradiation
  3. Measurement of calcium deposition
  4. Alizarin red S staining
  5. Alkaline phosphatase(ALP) assay
  6. Total RNA isolation and quantitative RT-PCR
  7. Cell viability
  8. Statistical analysis
 III. RESULTS
  1. Bone mineralization and ALP activity
  2. mRNA expression of MC3T3-E1 differentiation-related genes
  3. Determination of PMA concentration
  4. Bone mineralization on PMA-treatedMC3T3-E1 cells
  5. mRNA expression in PMA-treatedMC3T3-E1 cells
 IV. DISCUSSION
 V. REFERENCES

• 권혁일(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Hyuk Il Kwon
• 김상우(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Sang Woo Kim
• 김서연(Department of Dental Hygiene, Songwon College University) | Seo Yune Kim
• 김옥준(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Ok Joon Kim
• 최홍란(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Hong Ran Choi correspondence
• 고영종(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Young Jong Ko
• 김인애(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | In Ae Kim
• 김지선(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Ji Sun Kim
• 원재웅(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Jae Woong Won
• 임원봉(Dental Research Institute, Department of Oral Pathology, School of Dentistry, Chonnam National University) | Won Bong Lim